U.S. patent application number 13/871283 was filed with the patent office on 2014-02-06 for synthetic cell platforms and methods of use thereof.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is The Regents of the University of California. Invention is credited to Patrick Sean Daugherty, Kevin Edward Healy, Lauren Little, David V. Schaffer.
Application Number | 20140037696 13/871283 |
Document ID | / |
Family ID | 39789191 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140037696 |
Kind Code |
A1 |
Schaffer; David V. ; et
al. |
February 6, 2014 |
Synthetic Cell Platforms and Methods of Use Thereof
Abstract
The present invention provides synthetic cell platforms. The
synthetic cell platforms can be used for culturing cells in vitro.
The synthetic cell platforms can also be implanted together with
bound cells into an individual. The present invention provides
methods of using the platforms to provide cells or progeny of such
cells for use in various applications, including clinical
applications; and methods of use of the platforms to introduce
cells into an individual.
Inventors: |
Schaffer; David V.;
(Danville, CA) ; Healy; Kevin Edward; (Moraga,
CA) ; Little; Lauren; (Oakland, CA) ;
Daugherty; Patrick Sean; (Santa Barbara, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
39789191 |
Appl. No.: |
13/871283 |
Filed: |
April 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12532098 |
Mar 18, 2010 |
8501905 |
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PCT/US2008/003811 |
Mar 21, 2008 |
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13871283 |
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60919640 |
Mar 22, 2007 |
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Current U.S.
Class: |
424/400 ;
424/93.7; 435/174; 435/7.32; 514/21.4; 514/21.5; 530/326;
530/327 |
Current CPC
Class: |
C12N 2533/90 20130101;
G01N 33/5073 20130101; G01N 33/542 20130101; C12N 5/0606 20130101;
C12N 2533/50 20130101; C12N 2501/115 20130101; C12N 2500/99
20130101; A61P 43/00 20180101; C12N 2501/15 20130101; C07K 17/00
20130101 |
Class at
Publication: |
424/400 ;
435/7.32; 530/326; 435/174; 424/93.7; 530/327; 514/21.5;
514/21.4 |
International
Class: |
C07K 17/00 20060101
C07K017/00 |
Claims
1. A method of identifying a peptide that binds a cell surface
molecule present on a mammalian stem cell surface, the method
comprising: a) contacting a mammalian stem cell with a population
of bacterial cells, wherein each bacterial cell in the population
displays on its cell surface a different synthetic peptide, said
contacting resulting in a mammalian cell having bound to its
surface one or more bacteria, and unbound bacteria; and b)
separating the bound bacteria from the unbound bacteria, wherein
the synthetic peptide displayed by the bound bacterium is a peptide
that binds a cell surface molecule present on a mammalian stem cell
surface.
2.-4. (canceled)
5. A synthetic cell platform comprising: a) a synthetic substrate;
and b) a mammalian stem cell-binding peptide covalently linked to
the synthetic substrate, wherein the stem cell binding peptide
comprises the amino acid of any one of SEQ ID NOs:7-21, 23-51, and
60-103, and has a length of from about 5 amino acids to about 50
amino acids.
6. An implantable cell composition comprising: a) the synthetic
platform of claim 5; b) and a mammalian stem cell bound to the
peptide.
7. A method of culturing a mammalian stem cell in vitro, the method
comprising, contacting the mammalian stem cell with a synthetic
platform according to claim 5, in the absence of a feeder cell
layer and in the presence of a serum-free liquid medium.
8. The method of claim 7, wherein the mammalian stem cell is an
adult stem cell, an embryonic stem cell, or an induced pluripotent
stem cell.
9. The method of claim 7, wherein the synthetic platform promotes
self renewal of the stem cell.
10. The method of claim 7, wherein the synthetic platform promotes
differentiation of the stem cell.
11. A method of isolating a desired cell type or cell population,
the method comprising: a) contacting a mixed cell population
comprising the desired cell type or desired cell population with a
synthetic cell platform according to claim 5, forming a complex
comprising the desired cell type or desired cell population bound
to a peptide on the synthetic cell platform; and b) separating
unbound cells from the complex.
12. A method of enhancing survival of a cell implanted into a
recipient individual, the method comprising introducing into the
recipient individual an implantable composition of claim 6, wherein
the platform enhances survival of the cell in the recipient
individual.
13. A method of inducing in vivo differentiation of a stem cell
implanted into a recipient individual, the method comprising
introducing into the recipient individual an implantable
composition of claim 6, wherein the mammalian cell bound to the
cell platform is a stem cell, and wherein the platform induces
differentiation of the stem cell in vivo.
14. A method of promoting recruitment of a cell to a site in an
individual, the method comprising implanting into a site in the
individual a synthetic cell platform of claim 5, wherein the
covalently linked peptide promotes recruitment of the cell to the
site of implantation.
15. An implantable device comprising: a) a surface; and b)
synthetic cell platform of claim 5 coated onto the surface.
16. The device of claim 15, wherein the device is a stent.
17. The synthetic cell platform of claim 5, wherein the stem cell
binding peptide comprises the amino acid sequence WWCDMRGDSRCSG
(7C-24; SEQ ID NO:32), or DHKFGLVMLNKYAYAG (15-2; SEQ ID
NO:60).
18. The synthetic cell platform of claim 5, wherein the stem
cell-binding peptide is covalently linked to the synthetic
substrate at a density of from about 0.1 pmol/cm.sup.2 to about 100
pmol/cm.sup.2.
19. The synthetic cell platform of claim 5, wherein the stem
cell-binding peptide is covalently linked to the synthetic
substrate at a density of from about 1 pmol/cm.sup.2 to about 25
pmol/cm.sup.2.
20. The synthetic cell platform of claim 5, wherein the synthetic
substrate is an interpenetrating polymer network, a synthetic
hydrogel, a semi-interpenetrating polymer network, or a
thermo-responsive polymer.
21. The synthetic cell platform of claim 5, wherein the synthetic
substrate comprises a co-polymer of polyacrylamide and
poly(ethylene glycol).
22. The synthetic cell platform of claim 5, wherein the stem cell
is an embryonic stem cell, a neural stem cell, a hematopoietic stem
cell, a mesenchymal stem cell, or an induced pluripotent stem
cell.
23. The synthetic cell platform of claim 5, further comprising a
mammalian stem cell bound to the peptide.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/919,640, filed Mar. 22, 2007, which
application is incorporated herein by reference in its
entirety.
BACKGROUND
[0002] Human embryonic stem cells (hESCs) have potential as sources
of cells for the treatment for disease and injury (e.g. tissue
engineering and reconstruction, diabetes, Parkinson's Disease,
leukemia, congestive heart failure, etc.). Features that are
important for successful integration of hESC into such therapies
include: expansion of hESCs without differentiation (i.e.,
self-renewal), differentiation of hESCs into a specific cell type
or collection of cell types, and functional integration of hESCs or
their progeny into existing tissue. Current ex vivo culture systems
for hESCs include mouse and human feeder cell layers, media
conditioned by feeder cells, or serum-free conditions with complex
extracellular matrix proteins. Such systems pose a number of
problems, including poorly characterized environmental signals, the
transmission of pathogens to hESCs, the transfer of (and
"contamination" with) immunogenic epitopes to hESCs leading to
rejection after engraftment, poor availability of large-scale
supplies of reproducibly high quality purified proteins, and
limitations on the ability to scale-up to a clinical process for
the treatment of thousands or even millions of patients. In
addition, the grafting of hESCs or their differentiated progeny in
vivo for tissue repair often suffers from poor cell viability.
Therefore, improved platforms are needed for enhancing the survival
of implanted cells.
[0003] There is a need in the art for improved culture systems and
methods for generating stem cells, e.g., hESCs, and/or progeny
thereof for clinical use.
LITERATURE
[0004] U.S. Pat. No. 7,157,275; U.S. Patent Publication No.
2007/0026518; U.S. Pat. No. 5,863,650; U.S. Patent Publication No.
2004/0001892; and U.S. Patent Publication No. 2007/0099247.
SUMMARY OF THE INVENTION
[0005] The present invention provides synthetic cell platforms. The
synthetic cell platforms can be used for culturing cells in vitro.
The synthetic cell platforms can also be implanted together with
bound cells into an individual. The present invention provides
methods of using the platforms to provide cells or progeny of such
cells for use in various applications, including clinical
applications; and methods of use of the platforms to introduce
cells into an individual.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is a schematic depiction of a biomimetic
interpenetrating polymer network.
[0007] FIG. 2 depicts multi-speed adhesion assay results.
[0008] FIG. 3 depicts bright field images of neural stem cells
grown on peptide-modified IPNs or laminin-I in proliferating media
conditions.
[0009] FIGS. 4A-D depict features of neural stem cells cultured in
vitro.
[0010] FIG. 5 depicts isolation of cell-specific peptides using
bacterial display.
[0011] FIG. 6a depicts peptides; and FIGS. 6b and 6c depict
isolation of neural stem cell binding bacterial clones. The
peptides depicted in FIG. 6a are: WWCDMRGDSRCSG (SEQ ID NO:32);
YMCMSRGDATCDV (SEQ ID NO:17); QCCQLRGDAVCNC (SEQ ID NO:26);
WVCNKLGVYACEY (SEQ ID NO:28); LECTERGDFNCFV (SEQ ID NO:20); AND
ESCWYQIMYKCAN (SEQ ID NO:15).
[0012] FIGS. 7A-F depict morphology and Oct4 immunofluorescence of
hESCs cultured in vitro.
[0013] FIGS. 8A-G depict analysis of phosphorylated/activated
Akt.
[0014] FIG. 9 is a schematic depiction of a BKAR sensor.
[0015] FIG. 10 depicts library population binding to neural stem
cells after each round of selection.
[0016] FIG. 11 depicts surface peptide concentration of fluorescent
peptides on the interpenetrating network (IPN).
[0017] FIG. 12 depicts initial cell attachment on peptide-grafted
IPNs.
[0018] FIG. 13 depicts cell proliferation on peptide-grafted IPNs
after 5 days.
[0019] FIG. 14 depicts cell proliferation after 5 days based on
surface peptide concentration.
DEFINITIONS
[0020] As used herein, the term "stem cell" refers to an
undifferentiated cell that can be induced to proliferate. The stem
cell is capable of self-maintenance or self-renewal, meaning that
with each cell division, one daughter cell will also be a stem
cell. Stem cells can be obtained from embryonic, post-natal,
juvenile, or adult tissue. Stem cells can be pluripotent or
multipotent. The term "progenitor cell," as used herein, refers to
an undifferentiated cell derived from a stem cell, and is not
itself a stem cell. Some progenitor cells can produce progeny that
are capable of differentiating into more than one cell type.
[0021] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment," as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease.
[0022] The terms "individual," "subject," "host," "recipient," and
"patient," used interchangeably herein, refer to a mammal,
including, but not limited to, rodents (e.g., rats, mice),
non-human primates, humans, canines, felines, ungulates (e.g.,
equines, bovines, ovines, porcines, caprines), etc.
[0023] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0024] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0025] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0026] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a stem cell" includes a plurality of such
stem cells and reference to "the cell culture platform" includes
reference to one or more cell culture platforms and equivalents
thereof known to those skilled in the art, and so forth. It is
further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0027] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION
[0028] The present invention provides synthetic cell platforms. The
synthetic cell platforms can be used for culturing cells in vitro.
The synthetic cell platforms can also be implanted together with
bound cells into an individual. The present invention provides
methods of using the platforms to provide cells or progeny of such
cells for use in various applications, including clinical
applications; and methods of use of the platforms to introduce
cells into an individual.
Methods of Identifying Peptide Ligands
[0029] The present invention provides methods of identifying
peptide ligands of mammalian cell surface molecule (e.g., a cell
surface macromolecule such as a protein, a glycoprotein, etc.). The
methods generally involve contacting a mammalian cell in vitro with
a population of bacteria comprising individual bacteria, each of
which displays on its surface a different heterologous peptide,
forming a mixed mammalian cell population comprising mammalian
cells bound to one or more bacteria and unbound bacterial cells;
and separating the bound from the unbound bacterial cells. Binding
of a mammalian cell to a bacterium indicates that the bacterium
displays on its cell surface a peptide that binds to a mammalian
cell surface molecule. The heterologous peptide displayed by the
bacterium is considered a candidate cell surface-binding peptide
for use in a subject synthetic cell platform. Methods of generating
a peptide display library include, e.g., a method as described in
U.S. Patent Publication No. 2007/0099247, which is incorporated
herein by reference.
[0030] A peptide-displaying bacterium displays a peptide at a
density of from about 10.sup.3 to about 10.sup.5 peptides per
bacterial cell, e.g., at a density of from about 10.sup.3 to about
5.times.10.sup.3, from about 5.times.10.sup.3 to about 10.sup.4,
from about 10.sup.4 to about 5.times.10.sup.4, or from about
5.times.10.sup.4 to about 10.sup.5, or more, peptide molecules per
cell. Each bacterium in a peptide-displaying bacterial population
(or "library") displays a different peptide. A peptide-displaying
bacterial library can display two, three, four, five, six, seven,
eight, nine, ten, from 10 to 25, from 25 to 50, from 50 to 100,
from 10.sup.2 to 10.sup.4, from 10.sup.4 to 10.sup.6, from 10.sup.6
to 10.sup.8, from 10.sup.8 to 10.sup.9, or from 10.sup.9 to about
10.sup.10, or more, different peptides, each present on the surface
of different bacteria in the library population.
[0031] A peptide displayed by a peptide-displaying bacterium is
"heterologous," e.g., the peptide is one that is not normally
synthesized by the bacterium. Each heterologous peptide can have a
length of from about 5 amino acids to about 50 amino acids, e.g.,
from about 5 amino acids to about 10 amino acids, from about 10
amino acids to about 15 amino acids, from about 15 amino acids to
about 20 amino acids, from about 20 amino acids to about 25 amino
acids, from about 25 amino acids to about 30 amino acids, from
about 30 amino acids to about 35 amino acids, from about 35 amino
acids to about 40 amino acids, from about 40 amino acids to about
45 amino acids, or from about 45 amino acids to about 50 amino
acids.
[0032] In some embodiments, the heterologous peptide is displayed
as a fusion protein of a bacterial protein, e.g., a fusion protein
comprising a bacterial protein (e.g., a protein that the bacterium
normally synthesizes) fused in-frame to a heterologous peptide. The
heterologous peptide can be fused to the amino terminus of the
bacterial protein, to the carboxyl-terminus of the bacterial
protein, or at an internal site of the bacterial protein. The
fusion protein is synthesized by the bacterium such that the
heterologous peptide is displayed on the surface of the bacterium,
e.g, a surface that is accessible for binding by a mammalian cell
when the bacterium and the mammalian cell are brought into contact
in vitro.
[0033] In some embodiments, the heterologous peptide is displayed
as a fusion protein with an outer membrane protein (OMP) of a
bacterium. In some embodiments, the heterologous peptide is a
C-terminal or N-terminal fusion protein with a circularly permuted
variant of outer membrane protein X (CPX). CPX is described in,
e.g., Rice et al. (2006) Protein Science 15:825-836. See also, U.S.
Patent Publication No. 2007/0099247.
[0034] In some embodiments, the peptide-displaying bacteria are
genetically modified to produce a fluorescent protein, e.g., the
peptide-displaying bacteria are genetically modified by
introduction into the bacteria of a nucleic acid comprising a
nucleotide sequence encoding a fluorescent protein, where the
nucleic acid is, e.g., an expression vector that provides for
expression of the nucleotide sequence in the bacteria. Suitable
fluorescent proteins include, but are not limited to, a green
fluorescent protein (GFP; Chalfie, et al., Science
263(5148):802-805 (Feb. 11, 1994); an enhanced GFP (EGFP), e.g.,
Genbank Accession Number U55762); a blue fluorescent protein (BFP;
Stauber, R. H. Biotechniques 24(3):462-471 (1998); Heim, R. and
Tsien, R. Y. Curr. Biol. 6:178-182 (1996)); a yellow fluorescent
protein (YFP); an enhanced yellow fluorescent protein (EYFP);
luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417 (1993));
a fluorescent protein as described in, e.g., WO 92/15673, WO
95/07463, WO 98/14605, WO 98/26277, WO 99/49019, U.S. Pat. No.
5,292,658, U.S. Pat. No. 5,418,155, U.S. Pat. No. 5,683,888, U.S.
Pat. No. 5,741,668, U.S. Pat. No. 5,777,079, U.S. Pat. No.
5,804,387, U.S. Pat. No. 5,874,304, U.S. Pat. No. 5,876,995, or
U.S. Pat. No. 5,925,558); a cyan fluorescent protein (CFP); a GFP
from a species such as Renilla reniformis, Renilla mulleri, or
Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle
et al. (2001) J. Protein Chem. 20:507-519; and any of a variety of
fluorescent and colored proteins from Anthozoan species, as
described in, e.g., Matz et al. (1999) Nature Biotechnol.
17:969-973; U.S. Patent Publication No. 2002/0197676, or U.S.
Patent Publication No. 2005/0032085; and the like.
[0035] Where the bacteria are genetically modified to produce a
fluorescent protein, bound mammalian cells (e.g., mammalian cells
to which are bound one or more peptide-displaying bacteria) are
separated from unbound mammalian cells using a
fluorescence-activated cell sorting (FACS) method.
[0036] In some embodiments, the mammalian cells are genetically
modified to produce a detectable signal upon binding of a
peptide-displaying bacterium that displays a peptide that activates
a cell signaling pathway. Signaling pathways include, but are not
limited to, phosphoinositide-3 kinase (PI 3-kinase),
mitogen-activated protein kinase (MAPK), phospholipase C (PLC),
protein kinase C (PKC), protein kinase A (PICA), protein kinase G
(PKG), Sonic hedgehog (Shh), Wnt, Notch, Jak/STAT, and
calcium/calmodulin dependent kinase (Cam kinase). The assay used to
detect activation of a cell signaling pathway can depend, e.g., on
the mode of transmission of the signal and/or the nature of the
components of the signaling pathway. For example, in some
embodiments, detecting activation of a signaling pathway involves
detecting activity of a kinase involved in the signaling pathway.
In other embodiments, detecting activation of a signaling pathway
involves detecting a change in intracellular calcium concentration
([Ca.sup.2+].sub.i). In other embodiments, detecting activation of
a signaling pathway involves detecting relocalization of one or
more molecules in the cell interior (e.g., relocalization of a
protein from the cytoplasm to the nucleus, and the like). In some
embodiments, a fluorescence resonance energy transfer (FRET)-based
assay is used.
[0037] As one non-limiting example, to detect activation of a
signaling pathway, the activity of a kinase can be detected, e.g.,
phosphorylation of Akt by PDK1; and the like. In some embodiments,
a FRET-based assay is used. In some embodiments, a mammalian cell
is genetically modified with a nucleic acid comprising a nucleotide
sequence that encodes, in order from amino terminus to carboxyl
terminus, a first member of a FRET pair (e.g., a FRET donor), a
phosphoamino acid binding domain, an amino acid sequence that is
specifically recognized and phosphorylated by the kinase, and a
second member of a FRET pair (e.g., a FRET acceptor). Various
fluorescent proteins can serve as FRET pairs. For example, CFP has
an excitation maximum at 433 nm and an emission maximum at 476 nm,
and can be used as a donor fluorophore in combination with a YFP as
an acceptor (emission maximum at 527 nm). As another example, a BFP
can be used as a donor fluorophore in combination with a GFP as the
acceptor, or a CFP can be used as the donor fluorophore in
combination with a YFP as the acceptor.
[0038] In some embodiments, the first member of the FRET pair is a
CFP; and the second member of the FRET pair is a YFP. In some
embodiments, the phosphoamino acid binding domain is an FHA2
phosphothreonine-binding domain. In some embodiments, the amino
acid sequence that is specifically recognized and phosphorylated by
the kinase is a consensus PKB phosphorylation sequence RKRDRLGTLGI
(SEQ ID NO:1), where the underlined T is the phospho-acceptor
residue. In some embodiments, the first and/or the second member of
the FRET pair is a mutant CFP or a mutant YFP, e.g., a CFP or a YFP
with a A206K mutation that renders the protein monomeric. See,
e.g., Jones et al. (1991) Proc. Natl. Acad. Sci. U.S.A.
88:4171-4175; and Kunkel et al. (2005) J. Biol. Chem.
280:5581-5587. In some embodiments, the fluorescent protein is a
mutant as described in Nguyen and Daugherty (2005) Nat. Biotechnol.
23:355-360.
[0039] FRET is phenomenon known in the art wherein excitation
energy of one fluorescent dye is transferred to another without
emission of a photon. A FRET pair consists of a donor fluorophore
and an acceptor fluorophore (where the acceptor fluorophore may be
a quencher molecule). The fluorescence emission spectrum of the
donor and the fluorescence absorption spectrum of the acceptor must
overlap, and the two molecules must be in close proximity. The
distance between donor and acceptor at which 50% of donors are
deactivated (transfer energy to the acceptor) is defined by the
Forster radius, which is typically 10-100 angstroms. Changes in the
fluorescence emission spectrum comprising FRET pairs can be
detected, indicating changes in the number of that are in close
proximity (i.e., within 100 angstroms of each other). This will
typically result from the binding or dissociation of two molecules,
one of which is labeled with a FRET donor and the other of which is
labeled with a FRET acceptor, wherein such binding brings the FRET
pair in close proximity. Suitable FRET acceptors and donors (e.g.,
first and second members of a FRET pair) include the
above-mentioned fluorescent proteins, e.g, cyan fluorescent protein
(CFP), yellow fluorescent protein, red fluorescent protein, green
fluorescent protein, and the like.
[0040] Binding of such molecules will result in an increased
fluorescence emission of the acceptor and/or quenching of the
fluorescence emission of the donor. As an example, for detecting
activation of Akt, a donor cyan fluorescent protein (CFP) and
acceptor yellow fluorescent protein (YFP) with an intervening Akt
substrate peptide yields FRET signal changes with Akt signaling and
phosphorylation. This system can be adapted to provide for analysis
of activation of any of a number of kinases. In some embodiments, a
CFP mutant (e.g., A206K mutant) and a YFP mutant (e.g., A206K
mutant) are used, which mutants are described in, e.g., Nguyen and
Daugherty (2005) Nat. Biotechnol. 23:355-360; and Shaner et al.
(2005) Nat. Methods 2:905-909. Akt is also known in the art as
protein kinase B (PKB). A PKB sensor (also referred to as a B
kinase activity reporter, or BKAR) is depicted schematically in
FIG. 9. An expression construct comprises a nucleotide sequence
encoding CFP, the FHA2 domain of Rad53p, a consensus PKB
phosphorylation sequence (e.g., RKRDRLGTLGI (SEQ ID NO:1), where
the underlined T is the phospho-acceptor residue), and YFP. See,
e.g., Kunkel et al. (2005) J. Biol. Chem. 280:5581-5587. In the
presence of active PKB, BKAR undergoes a conformational change,
such that no signal is produced.
Peptides
[0041] The present invention provides peptides, e.g., isolated
peptides or synthetic peptides, identified by a subject screening
method, as described above. The peptides are useful for generating
synthetic cell platforms, as described below. Peptides include, but
are not limited to, integrin-binding peptides; peptides that bind a
non-integrin adhesion receptor on the surface of a mammalian cell;
peptides that activate a signaling pathway in a mammalian cell; and
the like.
[0042] A subject peptide can have a length of from about 5 amino
acids to about 50 amino acids, e.g., from about 5 amino acids to
about 10 amino acids, from about 10 amino acids to about 15 amino
acids, from about 15 amino acids to about 20 amino acids, from
about 20 amino acids to about 25 amino acids, from about 25 amino
acids to about 30 amino acids, from about 30 amino acids to about
35 amino acids, from about 35 amino acids to about 40 amino acids,
from about 40 amino acids to about 45 amino acids, or from about 45
amino acids to about 50 amino acids.
[0043] Exemplary peptides are show in Tables 1-4, below, and also
in Table 5 in Example 5.
TABLE-US-00001 TABLE 1 Receptor Peptide Sequence Target Protein
Source CGGNGEPRGDTYRAY (bsp-RGD(15) .alpha..sub..nu..beta..sub.3
Bone sialoprotein, (SEQ ID NO: 2) vitronectin C*EPRGDTYRAYG*
[c-bsp-RGD(12)] .alpha..sub..nu..beta..sub.3 Bone sialoprotein,
(SEQ ID NO: 3) vitronectin VSWFSRHRYSPFAVS
.alpha..sub.6.beta..sub.1 Laminin-1 (.alpha.) (SEQ ID NO: 4)
C*TRKKHDNAQC* .alpha..sub.2.beta..sub.1 Coll(I), (SEO ID NO: 5)
Laminin-1 (.alpha.) KQNCLSSRASRGCVRNLRLSR .alpha..sub.3.beta..sub.1
Laminin-1 (.alpha.) (SEO ID NO: 6)
TABLE-US-00002 TABLE 2 Clone Peptide Sequence 15-2 DHKFGLVMLNKYAYAG
(SEQ ID NO: 7) 15-6 LEDAMGWALSWGHIW (SEQ ID NO: 8) 15-16
SDWSVLLSCERWYCI (SEQ ID NO: 9) 15-32 RRELVRMTDWVWVSG (SEQ ID NO:
10) 15-50 GFVLVWSYTCRCWGK (SEQ ID NO: 11) 15-52 ESGLKVMCMKYYCMA
(SEQ ID NO: 12) 15-59 DLCTYGHLWLGNGRP (SEQ ID NO: 13)
TABLE-US-00003 TABLE 3 Clone Peptide sequence 7C-1 WYCFRENKYVCVM
(SEQ ID NO: 14) 7C-2 ESCWYQIMYKCAN (SEQ ID NO: 15) 7C-3
WFCLLGRSAYCVR (SEQ ID NO: 16) 7C-4 YMCMSRGDATCDV (SEQ ID NO: 17)
7C-5 IWCGSRFGCWCKP (SEQ ID NO: 18) 7C-6 GECFYYVMNTCVW (SEQ ID NO:
19) 7C-7 LECTERGDFNCFV (SEQ ID NO: 20) 7C-8 WLCLDKNCMACVW (SEQ ID
NO: 21) 7C-9 KLCCFDKGYYCMR (SEQ ID NO: 22) 7C-11 LCCESYICALCHY (SEQ
ID NO: 23) 7C-12 FWCIRGEYWVCDR (SEQ ID NO: 24) 7C-14 LNCAMYNACIW
(SEQ ID NO: 25) 7C-15 QCCQLRGDAVCNC (SEQ ID NO: 26) 7C-17
WLCKGSNKYMCEW (SEQ ID NO: 27) 7C-19 WVCNKLGVYACEY (SEQ ID NO: 28)
7C-20 WVCIWERFKSCNE (SEQ ID NO: 29) 7C-21 WNCIKGSSWACVW (SEQ ID NO:
30) 7C-22 WMCSGVQPNACVW (SEQ ID NO: 31) 7C-24 WWCDMRGDSRCSG (SEQ ID
NO: 32)
TABLE-US-00004 TABLE 4 Clone Peptide Sequence Co-1 SLCAAYNRWACIW
(SEQ ID NO: 33) Co-2 WSCPKVNQYACFW (SEQ ID NO: 34) Co-3
GGCRWYAKWVCVW (SEQ ID NO: 35) Co-5 WDCGKKNAWMCIW (SEQ ID NO: 36)
Co-8 WTWESAFAGRWEVGD (SEQ ID NO: 37) Co-9 SKCWGWTPYYCVA (SEQ ID NO:
38) Co-10 WRCLGDGYHACVR (SEQ ID NO: 39) Co-11 LECPGESKYYCIY (SEQ ID
NO: 40) Co-12 WVCLWRHRGDCSI (SEQ ID NO: 41) Co-13 STCSWVSSYVCIM
(SEQ ID NO: 42) Co-15 WVCNDLIHEYCVW (SEQ ID NO: 43) Co-16
QGCAFVTYWACIF (SEQ ID NO: 44) Co-17 WECAEESKFWCVF (SEQ ID NO: 45)
Co-18 WWCKKPEYWYCIW (SEQ ID NO: 46) Co-19 WQCGRFWCIHCLW (SEQ ID NO:
47) Co-20 RLCCWKTQYFCEI (SEQ ID NO: 48) Co-21 MYCERDSKYWCIH (SEQ ID
NO: 49) Co-22 VWCGMFGKRRCVT (SEQ ID NO: 50) Co-23 LVCNRQNPWVCYI
(SEQ ID NO: 51)
[0044] Further exemplary peptides are shown in Table 5, in Example
5. In some embodiments, a subject peptide comprises an amino acid
sequence as shown in Tables 1-5 (e.g., a subject peptide comprises
an amino acid sequence as set forth in any one of SEQ ID NOs:2-51,
and 60-103). For example, any of the peptide sequences depicted in
Tables 1-5 (e.g., SEQ ID NOs: 2-51, and 60-103) can include one or
more additional amino acids on the amino- and/or carboxyl-terminus
of the peptide. In some embodiments, the peptide includes a linker
that provides for linkage to a matrix or synthetic substrate, as
described in more detail below. Also included are peptides that
include one or more amino acid substitutions compared to a peptide
sequence depicted in Tables 1-5 (e.g., any one of SEQ ID NOs: 2-51,
and 60-103). In some embodiments, a subject peptide comprises an
amino acid sequence of any one of SEQ ID NOs:7-103. In some
embodiments, a subject peptide comprises an amino acid sequence of
any one of SEQ ID NOs:7-51 and 60-103.
[0045] In some embodiments, a peptide comprising one or more of the
following sequences is specifically excluded: RGD, FHRRIKA, PRRARV,
REDV, DEGA, YIGSR, IKVAV, PHSRN, and KGD, and cyclic variants
thereof. In some embodiments, one or more of the peptides shown in
Table 1 (SEQ ID NOs:2-6) are specifically excluded. In some
embodiments, a peptide comprising one or more of SEQ ID NOs:52-59
is specifically excluded.
[0046] Peptides can be synthesized using standard methods for
chemical synthesis of a peptide. Peptides can also be synthesized
recombinantly, using standard methods.
[0047] In some embodiments, a subject peptide, when contacted with
a stem cell, induces differentiation of the stem cell in vitro
and/or in vivo. In other embodiments, a subject peptide, when
contacted with a stem cell, promotes growth of the stem cell
without inducing differentiation of the stem cell, in vitro and/or
in vivo. In some embodiments, a subject peptide, when contacted
with a stem cell, self-renewal of the stem cell in vitro and/or in
vivo. In other embodiments, a subject peptide promotes growth
(proliferation) of a differentiated cell in vitro and/or in
vivo.
Synthetic Cell Platforms
[0048] The present invention provides a synthetic cell platform
that comprises a synthetic substrate and a cell surface-binding
peptide. In some embodiments, the cell surface-binding peptide is a
peptide identified by a subject method. A subject synthetic cell
culture platform is useful for culturing cells in vitro, where the
synthetic cell culture platform promotes growth and/or
differentiation of a cell in in vitro culture. A subject synthetic
cell culture platform can also be implanted into an individual
along with cultured cells. As such, in some embodiments, a subject
synthetic cell culture platform is also an implantable cell
matrix.
[0049] Suitable cell surface-binding peptides include any of the
peptides depicted in Tables 1-5 (SEQ ID NOs:2-51, and 60-103), or
variants thereof. In some embodiments, suitable cell
surface-binding peptides include any of the peptides depicted in
Tables 2-5 (SEQ ID NOs:7-51 and 60-103), or variants thereof,
including cyclic variants. A cell surface-binding peptide can be
covalently linked to the synthetic substrate. A subject synthetic
cell platform can comprise a single type ("species") of
cell-binding peptide, e.g., where peptides of a given type or
species all have the same amino acid sequence; or can include two
or more types of cell-binding peptides, e.g., can comprise peptides
of two or more (e.g., two, three, four, five, or more) different
amino acid sequences that target the same cell surface receptor or
class of cell surface receptors, or that target different cell
surface receptors. For example, a subject cell platform can
comprise a single peptide species having an amino acid sequence,
where the peptide species binds a single type of cell. As another
example, a subject cell platform can comprise a first peptide
species having a first amino acid sequence, where the first peptide
species binds a cell of a first cell type; a second peptide species
having a second amino acid sequence that is different from the
first amino acid sequence, where the second peptide species binds a
cell of a second cell type that is different from the first cell
type; etc.
[0050] A cell-binding peptide is bound to a synthetic substrate at
a density of from about 0.01 pmol/cm.sup.2 to about 100
pmol/cm.sup.2, e.g., from about 0.01 pmol/cm.sup.2 to about 0.1
pmol/cm.sup.2, from about 0.1 pmol/cm.sup.2 to about 1
pmol/cm.sup.2, from about 1 pmol/cm.sup.2 to about 10
pmol/cm.sup.2, from about 10 pmol/cm.sup.2 to about 25
pmol/cm.sup.2, from about 25 pmol/cm.sup.2 to about 50
pmol/cm.sup.2, or from about 50 pmol/cm.sup.2 to about 100
pmol/cm.sup.2.
[0051] Suitable synthetic substrates include polymeric materials.
Suitable polymeric materials include, e.g., materials described in,
e.g., U.S. Patent Publication No. 2007/0026518, U.S. Patent
Publication No. 2004/0001892, and U.S. Pat. No. 5,863,650, each of
which is incorporated by reference herein for disclosure relating
to synthetic substrates. For example, suitable substrates include
interpenetrating polymer networks (IPNs); a synthetic hydrogel; a
semi-interpenetrating polymer network (sIPN); a thermo-responsive
polymer; and the like. For example, in some embodiments, a
synthetic substrate comprises a co-polymer of polyacrylamide and
poly(ethylene glycol) (PEG). In some embodiments, the synthetic
substrate comprises a co-polymer of polyacrylamide and PEG, and
further comprises acrylic acid.
[0052] A subject synthetic cell platform can be in any of a variety
of forms, e.g., a 3-dimensional form (e.g., suitable for implanting
into a tissue; or suitable as a synthetic tissue, for implanting
into a recipient individual); a flat surface (e.g., suitable for
coating onto the surface of an implantable device, such as an
intravascular stent, an artificial joint, a scaffold, etc.); and
the like.
[0053] In some embodiments, a subject synthetic cell platform
comprises one or more mammalian cells bound thereto; and is useful
for, e.g., introducing the cells into a recipient individual (e.g.,
a mammalian subject). In other embodiments, a subject synthetic
cell platform is contacted in vitro with one or more mammalian
cells; and the cells are cultured with the synthetic cell platform
in vitro. In other embodiments, mammalian cells are cultured with a
subject synthetic cell platform in vitro, then the cultured cells,
which remain associated with the platform, and introduced into a
recipient individual.
[0054] In some embodiments, a subject synthetic cell platform
without any bound mammalian cells is implanted into a recipient
individual, where the synthetic cell platform comprises one or more
species of peptides that recruit one or more cell types to the site
of implantation.
[0055] In some embodiments, a subject synthetic cell platform
without any bound mammalian cells is coated onto the surface of an
implantable device, forming a coated device. When the coated device
is implanted into a recipient individual, the peptide present in
the cell platform coated onto the device recruits one or more cell
types (e.g., endogenous cells present in the individual) to the
site of implantation. A subject synthetic cell platform can be
coated onto a device comprising any of a variety of materials,
including, but not limited to, plastics, including any
biocompatible plastic; glass, e.g., silicon dioxide, and the like;
metals, e.g., titanium; metal alloys, e.g., nickel titanium, etc.;
or any other material that can be implanted into a recipient
subject (e.g., a human) without causing substantial adverse
effects. In some embodiments, e.g., a subject synthetic cell
platform is coated onto a stent, where the peptide in the synthetic
cell platform recruits endothelial precursors.
[0056] The present invention thus provides an implantable device
comprising a surface and a subject synthetic cell platform coated
onto the surface, forming a coated implantable device.
Stem Cells and Progenitor Cells
[0057] Cells that are suitable for culturing on a subject cell
platform and/or including with a subject cell culture platform
(e.g., to form an implantable cell composition) include, but are
not limited to, stem cells, e.g., hematopoietic stem cells,
embryonic stem cells, mesenchymal stem cells, neural stem cells,
epidermal stem cells, endothelial stem cells, gastrointestinal stem
cells, liver stem cells, cord blood stem cells, amniotic fluid stem
cells, skeletal muscle stem cells, smooth muscle stem cells (e.g.,
cardiac smooth muscle stem cells), pancreatic stem cells, olfactory
stem cells, hematopoietic stem cells, induced pluripotent stem
cells; and the like; as well as differentiated cells that can be
cultured in vitro and used in a therapeutic regimen, where such
cells include, but are not limited to, keratinocytes, adipocytes,
cardiomyocytes, pancreatic islet cells, retinal cells, and the
like. The cell that is used will depend in part on the nature of
the disorder or condition to be treated.
[0058] Suitable human embryonic stem (ES) cells include, but are
not limited to, any of a variety of available human ES lines, e.g.,
BG01 (hESBGN-01), BG02 (hESBGN-02), BG03 (hESBGN-03) (BresaGen,
Inc.; Athens, Ga.); SA01 (Sahlgrenska 1), SA02 (Sahlgrenska 2)
(Cellartis AB; Goeteborg, Sweden); ES01 (HES-1), ES01 (HES-2), ES03
(HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6) (ES Cell
International; Singapore); UC01 (HSF-1), UC06 (HSF-6) (University
of California, San Francisco; San Francisco, Calif.); WA01 (H1),
WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14) (Wisconsin Alumni
Research Foundation; WARF; Madison, Wis.). Cell line designations
are given as the National Institutes of Health (NIB) code, followed
in parentheses by the provider code. See, e.g., U.S. Pat. No.
6,875,607.
[0059] Suitable human ES cell lines can be positive for one, two,
three, four, five, six, or all seven of the following markers:
stage-specific embryonic antigen-3 (SSEA-3); SSEA-4; TRA 1-60; TRA
1-81; Oct-4; GCTM-2; and alkaline phosphatase.
[0060] Hematopoietic stem cells (HSCs) are mesoderm-derived cells
that can be isolated from bone marrow, blood, cord blood, fetal
liver and yolk sac. HSCs are characterized as CD34.sup.+ and
CD3.sup.-. HSCs can repopulate the erythroid,
neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic
cell lineages in vivo. In vitro, HSCs can be induced to undergo at
least some self-renewing cell divisions and can be induced to
differentiate to the same lineages as is seen in vivo. As such,
HSCs can be induced to differentiate into one or more of erythroid
cells, megakaryocytes, neutrophils, macrophages, and lymphoid
cells.
[0061] Neural stem cells (NSCs) are capable of differentiating into
neurons, and glia (including oligodendrocytes, and astrocytes). A
neural stem cell is a multipotent stem cell which is capable of
multiple divisions, and under specific conditions can produce
daughter cells which are neural stem cells, or neural progenitor
cells that can be neuroblasts or glioblasts, e.g., cells committed
to become one or more types of neurons and glial cells
respectively. Methods of obtaining NSCs are known in the art.
[0062] Mesenchymal stem cells (MSC), originally derived from the
embryonal mesoderm and isolated from adult bone marrow, can
differentiate to form muscle, bone, cartilage, fat, marrow stroma,
and tendon. Methods of isolating MSC are known in the art; and any
known method can be used to obtain MSC. See, e.g., U.S. Pat. No.
5,736,396, which describes isolation of human MSC.
[0063] An induced pluripotent stem (iPS) cells is a pluripotent
stem cell induced from a somatic cell, e.g., a differentiated
somatic cell. iPS cells are capable of self-renewal and
differentiation into cell fate-committed stem cells, including
neural stem cells, as well as various types of mature cells.
[0064] iPS cells can be generated from somatic cells, including
skin fibroblasts, using, e.g., known methods. iPS cells produce and
express on their cell surface one or more of the following cell
surface antigens: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E,
and Nanog. In some embodiments, iPS cells produce and express on
their cell surface SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E,
and Nanog. iPS cells express one or more of the following genes:
Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and
hTERT. In some embodiments, an iPS cell expresses Oct-3/4, Sox2,
Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT. Methods of
generating iPS are known in the art, and any such method can be
used to generate iPS. See, e.g., Takahashi and Yamanaka (2006) Cell
126:663-676; Yamanaka et. al. (2007) Nature 448:313-7; Wernig et.
al. (2007) Nature 448:318-24; Maherali (2007) Cell Stem Cell
1:55-70; Nakagawa et al. (2008) Nat. Biotechnol. 26:101; Takahashi
et al. (2007) Cell 131:861; Takahashi et al. (2007) Nat. Protoc.
2:3081; and Okita et al. (2007 Nature 448:313.
[0065] iPS cells can be generated from somatic cells (e.g., skin
fibroblasts) by genetically modifying the somatic cells with one or
more expression constructs encoding Oct-3/4 and Sox2. In some
embodiments, somatic cells are genetically modified with one or
more expression constructs comprising nucleotide sequences encoding
Oct-3/4, Sox2, c-myc, and Klf4. In some embodiments, somatic cells
are genetically modified with one or more expression constructs
comprising nucleotide sequences encoding Oct-4, Sox2, Nanog, and
LIN28.
Implantable Compositions
[0066] As noted above, a subject synthetic cell culture platform
can also be implanted into an individual along with one or more
mammalian cells. As such, in some embodiments, a subject synthetic
cell culture platform is also an implantable cell matrix. The
present invention provide an implantable composition comprising a
subject implantable cell matrix; and one or more mammalian cells
(e.g., a stem cell; a progenitor cell; an undifferentiated progeny
of a stem cell; a differentiated cell; a differentiated progeny of
a stem cell; etc.). A subject implantable composition comprises one
or more mammalian cells bound thereto, where "bound" refers to an
association of the cells with the cell platform. A mammalian cell
can be bound to the cell platform via interaction of a cell-binding
peptide in the platform with a cell surface molecule on the
mammalian cell. A subject implantable composition can comprise from
about 10 to about 10.sup.10 mammalian cells, e.g., from about 10 to
about 10.sup.2, from about 10.sup.2 to about 10.sup.4, from about
10.sup.4 to about 10.sup.5, from about 10.sup.5 to about 10.sup.6,
from about 10.sup.6 to about 10.sup.7, from about 10.sup.7 to about
10.sup.8, from about 10.sup.8 to about 10.sup.9, or from about
10.sup.9 to about 10.sup.10 mammalian cells.
Methods of Culturing a Cell
[0067] The present invention provides methods of culturing a
mammalian cell in vitro and/or in vivo. The methods generally
involving contacting a mammalian cell (e.g., a stem cell, a
progenitor cell, a differentiated progeny of a stem cell, a
differentiated cell, etc.) with a subject synthetic cell platform.
The contacting can occur in vitro and/or in vivo. In some
embodiments, the mammalian cell remains in contact with (e.g.,
bound to) the cell platform. In other embodiments, the mammalian
cell proliferates, and progeny of the bound cell are either in
contact with the cell platform, or are unbound and are present in
the cell culture medium (e.g., where the cell is cultured in
vitro), or are unbound and are integrated into the tissue of a host
(e.g., where the cell is introduced into an individual and is
therefore in vivo). The present method of cell culture provides for
culturing a cell in vitro without the need for a mouse or a human
feeder layer of cells, i.e., a subject cell culture system and
method is in the absence of a feeder layer of cells.
[0068] A suitable cell culture medium is used for in vitro culture,
where a suitable cell culture medium can include one or more of a
growth factor, vitamins, serum albumin (e.g., human serum albumin),
and the like. In some embodiments, the cell culture medium lacks
serum albumin.
[0069] In some embodiments, the peptide and the culture conditions
provide for self-renewal of a stem cell.
[0070] In other embodiments, the peptide and the culture conditions
provide for differentiation of a stem cell or progenitor cell into
a differentiated cell.
[0071] For example, a stem cell can be induced to differentiate
into a neuronal cell, an astrocyte, an oligodendrocyte, or a
neuronal precursor cell. Markers of interest include, but are not
limited to, .beta.-tubulin III or microtubule-associated protein 2
(MAP-2), characteristic of neurons; glial fibrillary acidic protein
(GFAP), present in astrocytes; galactocerebroside (GalC) or myelin
basic protein (MBP); characteristic of oligodendrocytes; Nestin or
Musashi, characteristic of neural precursors and other cells. A
mature neuronal cell can be characterized by an ability to express
one, two, three, four, five, six, seven, or all eight of: 160 kDa
neuro-filament protein, MAP2ab, glutamate, synaptophysin, glutamic
acid decarboxylase (GAD), tyrosine hydroxylase, GABA, and
serotonin. The differentiated cells forming neural progenitor
cells, neuron cells and/or glial cells can also be characterized by
expressed markers characteristic of differentiating cells. The in
vitro differentiated cell culture can be identified by detecting
molecules such as markers of the neuroectodermal lineage, markers
of neural progenitor cells, neuro-filament proteins, MAP2ab,
glutamate, synaptophysin, glutamic acid decarboxylase, GABA,
serotonin, tyrosine hydroxylase, .beta.-tubulin, .beta.-tubulin
III, GABA A.alpha.2 receptor, glial fibrillary acidic protein
(GFAP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), plp,
DM-20, O4, and NG-2 staining.
[0072] As another example, a stem cell can be induced to
differentiate into a hepatocyte. Hepatocyte lineage cells
differentiated from stem cells can display one, two, three, or
more, of the following markers: .alpha..sub.1-antitrypsin (AAT)
synthesis, albumin synthesis, asialoglycoprotein receptor (ASGR)
expression, absence of .alpha.-fetoprotein, evidence of glycogen
storage, evidence of cytochrome p450 activity, and evidence of
glucose-6-phosphatase activity.
[0073] As another example, a stem cell can be induced to
differentiate into a cardiomyocyte. In some embodiments,
differentiation into a cardiomyocyte is ascertained by detecting
cardiomyocyte-specific markers produced by the cell. For example,
the cardiomyocytes express cardiac transcription factors, sarcomere
proteins, and gap junction proteins. Suitable
cardiomyocyte-specific proteins include, but are not limited to,
cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3,
GATA-4, myosin heavy chain, myosin light chain-2a, myosin light
chain-2v, ryanodine receptor, and atrial natriuretic factor.
Whether a stem cell has differentiated into a cardiomyocyte can
also be determined by detecting responsiveness to pharmacological
agents such as .beta.-adrenergic agonists (e.g., isoprenaline),
adrenergic .beta.-antagonists (e.g., esmolol), cholinergic agonists
(e.g., carbochol), and the like. Whether a stem cell has
differentiated into a cardiomyocyte can also be determined by
detecting electrical activity of the cells. Electrical activity can
be measured by various methods, including extracellular recording,
intracellular recording (e.g., patch clamping), and use of
voltage-sensitive dyes. Such methods are well known to those
skilled in the art.
Cell Purification Methods
[0074] A subject synthetic cell platform can also be used to
isolated (e.g, purify) a desired cell population, a desired cell
type, etc. The present invention thus provides methods of isolating
a desired cell type or cell population, the method generally
involving contacting a mixed cell population that comprises a
desired cell population or a desired cell type, under conditions
that permit binding of the desired cell population or cell type to
bind to the synthetic cell platform; and separating bound from
unbound cells. Multiple rounds of the binding and separating steps
can be carried out. In addition, sequential binding and separating
steps can be carried out with two or more synthetic cell platforms,
each having covalently linked thereto a different peptide.
[0075] A subject method for isolating a desired cell population or
a desired cell type results in a selected cell population, e.g.,
the selected cell population comprises at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about 98%, at least about 99%, or
greater than 99%, of a desired cell type or cell population.
[0076] Desired cell populations/cell types can include, e.g., a
differentiated cell (e.g., a cardiomyocyte, a hepatocyte, a HSC
lineage cell, and the like); and a stem cell, e.g., an HSC, a
neural stem cell, an ESC, an MSC, an iPS, an adult stem cell, and
the like.
Therapeutic Methods
[0077] The present invention provides a method of treating a
disorder in an individual in need thereof, the method generally
involving implanting into the individual a subject implantable
composition. The present invention provides a method of increasing
survival of a cell implanted into a recipient individual, the
method generally involving implanting into the recipient individual
a cell bound to a subject cell platform. The present invention
provides a method of inducing or promoting in vivo differentiation
of a stem cell in a recipient individual, the method generally
involving implanting into the recipient individual a stem cell
bound to a subject cell platform.
[0078] A subject cell platform comprising a cell or progeny thereof
is introduced into an individual at a site that is appropriate to
the disorder being treated. Sites and modes of administration can
include, e.g., implantation (e.g., of a subject platform comprising
cardiomyocytes) into heart muscle; intravenous infusion (e.g., of a
subject platform comprising HSCs or HSC lineage cells);
implantation into the pancreas (e.g., of a subject platform
comprising pancreatic islet cells); intramuscular injection (e.g.,
of a subject platform comprising skeletal muscle or muscle
progenitor cells); intracranial implantation (e.g., of a subject
platform comprising neural cells or glial cells); intraocular
implantation (e.g., of a subject platform comprising neural cells
or glial cells); intrathecal implantation (e.g., of a subject
platform comprising neural cells or glial cells); and the like.
[0079] An "effective amount" of a subject synthetic cell platform
comprising cells or progeny is an amount that, when administered to
an individual in one or more doses, provides a therapeutic effect,
e.g., provides for introduction into the individual of a sufficient
number of cells to provide for a therapeutic effect. An effective
number of cells or progeny thereof can range from about 10.sup.3
cells to about 10.sup.9 cells, e.g., from about 10.sup.3 cells to
about 10.sup.4 cells, from about 10.sup.4 cells to about 10.sup.5
cells, from about 10.sup.5 cells to about 10.sup.6 cells, from
about 10.sup.6 cells to about 10.sup.7 cells, from about 10.sup.7
cells to about 10.sup.8 cells, or from about 10.sup.8 cells to
about 10.sup.9 cells.
[0080] For example, where a subject synthetic cell platform
comprises pancreatic islet cells, an effective amount of a subject
synthetic cell platform is an amount that includes pancreatic islet
cell in cell numbers that are effective to reduce a blood glucose
level in an individual by at least about 10%, at least about 15%,
at least about 20%, at least about 25%, at least about 30%, at
least about 40%, or at least about 50% when compared to the blood
glucose levels in the absence of the cells. In some embodiments,
effective number of HLA homozygous pancreatic islet cells is a
number that is effective to reduce blood glucose levels to a normal
range. Normal blood glucose levels are typically in the range of
from about 70 mg/dL to about 110 mg/dL before a meal (e.g., a
fasting blood glucose level); and less than 120 mg/dL 2 hours after
a meal.
[0081] A subject cell platform increases in vivo survival of a cell
bound thereto in a recipient individual. For example, a subject
implantable composition (a subject cell platform having bound
thereto a mammalian cell) provides for an at least about 10%, at
least about 20%, at least about 30%, at least about 50%, at least
about 75%, at least about 100% (or 2 fold), at least about 2.5
fold, at least about 5 fold, at least about 7.5 fold, at least
about 10-fold, or greater, increase in the length of time the cell
survives in the recipient individual, compared to the length of
time the cell would survive in the individual in the absence of the
cell platform.
[0082] In some embodiments, a subject cell platform increases in
vivo proliferation of a cell bound thereto in a recipient
individual. For example, a subject implantable composition (a
subject cell platform having bound thereto a mammalian cell)
provides for an at least about 10%, at least about 20%, at least
about 30%, at least about 50%, at least about 75%, at least about
100% (or 2 fold), at least about 2.5 fold, at least about 5 fold,
at least about 7.5 fold, at least about 10-fold, or greater,
increase in the number of cells generated by a single implanted
cell in the recipient individual, compared to the number cells
generated by the cell in the individual in the absence of the cell
platform.
[0083] In some embodiments, a subject cell platform increases in
vivo self renewal of a stem cell bound thereto in a recipient
individual. For example, a subject implantable composition (a
subject cell platform having bound thereto a mammalian cell)
provides for an at least about 10%, at least about 20%, at least
about 30%, at least about 50%, at least about 75%, at least about
100% (or 2 fold), at least about 2.5 fold, at least about 5 fold,
at least about 7.5 fold, at least about 10-fold, or greater,
increase in self renewal of an implanted stem cell in the recipient
individual, compared to the degree of self renewal of the stem cell
in the individual in the absence of the cell platform.
[0084] In some embodiments, a subject cell platform promotes in
vivo differentiation of a stem cell in a recipient individual. For
example, a subject implantable composition (a subject cell platform
having bound thereto a mammalian cell) provides for differentiation
of a stem cell in vivo in a recipient individual, where the stem
cell gives rise to one or more differentiated cell types in the
recipient individual.
Subjects Suitable for Treatment
[0085] Subjects suitable for treatment with a subject method
include individuals who have been diagnosed as having a blood cell
cancer (e.g., a leukemia); individuals who have been diagnosed with
AIDS; individuals with sickle cell anemia; individuals with an
immune disorder, e.g., an acquired immunodeficiency, a genetic
immunodeficiency; individuals with Type 1 diabetes; individuals
with a nervous system disorder such as Alzheimer's disease,
Parkinson's disease, Huntington's disease, Lou Gehrig's disease,
spinal cord injury, stroke, etc.; individuals with a liver disorder
such as hepatitis, cirrhosis, a metabolic disorder affecting the
liver or central nervous system (e.g., lysosomal storage disease);
individuals with a disorder of the cartilage or bone, e.g.,
individuals requiring joint replacement, individuals with
osteoarthritis, individuals with osteoporosis, etc.; individuals
with a cardiac disorder, e.g., myocardial infarction, coronary
artery disease, or other disorder resulting in ischemic cardiac
tissue; individuals with renal disorders, e.g., kidney failure
(e.g., individuals on kidney dialysis); individuals with skeletal
muscle disorders, such as muscular dystrophy; and individuals with
a lung disorder such as emphysema, pulmonary fibrosis, idiopathic
pulmonary fibrosis, etc.
EXAMPLES
[0086] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Example 1
Generation and Characterization of Synthetic Cell Platforms
[0087] This work was carried out with a synthetic material with
highly modular ligand-presentation capabilities. Furthermore, the
base material exhibits reduced non-specific interactions with
constituents of the biologic environment (e.g., proteins, lipids,
cells). As a first step toward creating such a platform technology,
interfacial interpenetrating polymer networks (IPNs) were developed
that are synthesized by sequential photoinitiated free-radical
polymerization of an ultrathin layer of polyacrylamide followed by
a secondary photoinitiation step using poly(ethylene glycol)-based
monomers to create the network (FIG. 1)..sup.29, 30, 41
Polyacrylamide, p(AAm), and poly(ethylene glycol), pEG, are
hydrophilic polymers that have demonstrated low protein, cell, and
bacterial binding characteristics, and are excellent materials for
precise control of cell behavior. These IPN layers based on pAAm
and pEG (p(AAm-co-EG)] have been grafted to both metal oxides
(e.g., SiO.sub.2, TiO.sub.2) and polymers (e.g., tissue culture
poly(styrene) and poly(ethylene terapthalate), i.e. PET).
Characterization of the IPNs by contact angle goniometry,
spectroscopic ellipsometry, x-ray photoelectron spectroscopy, and
static secondary ion mass spectrometry has confirmed the formation
of an interfacial IPN that resists protein adsorption and cell
adhesion..sup.29, 31, 51, 58 When the p(AAm-co-EG/AAc) IPN is
functionalized with the right bioactive ligand, it promotes cell
adhesion from a non-fouling platform, which is ideal for the cell
culture systems in this work.
[0088] FIG. 1. Schematic of the biomimetic interpenetrating polymer
network. An acrylamide layer is polymerized from the surface, and
subsequent polymerization of an interpenetrating polyethylene
glycol network makes the surface resistant to fouling/protein
adsorption. Finally, the surface can be decorated with bioactive
peptides or proteins for bioactive cell engagement.
[0089] Specific cell-binding surfaces were generated by developing
a high throughput system using the p(AAm-co-EG/AAc) IPN grafted to
polystyrene 24-well and 96-well plates to generate a library of
peptide modified surfaces of different types and densities
(>6500 independent surfaces were created)..sup.64 IPNs were
modified with both single ligands and ligand blends to study the
correlation between a simple metric, ligand-receptor adhesion
strength, and cell behavior (e.g. the extent of matrix
mineralization for osteoblasts in the cited work). The ligands
studied included cell-binding [CGGNGEPRGDTYRAY (bsp-RGD(15); SEQ ID
NO:2), CGGEPRGDTYRA (bsp-RGD(12); SEQ ID NO:52), CGPRGDTY
(bsp-RGD(8); SEQ ID NO:53), cyclic(CGPRGDTYG) (c-bsp-RGD(9); SEQ ID
NO:54), and CGPRGDTYG (bsp-RGD(9); SEQ ID NO:54)], heparin binding
(CGGFHRRIKA; SEQ ID NO:56), and collagen binding (CGGDGEAG; SEQ ID
NO:57) peptides, with the appropriate controls. Rat calvarial
osteoblast (RCO) adhesion to peptide-modified IPN polystyrene was
examined using a single-speed (600 RPM; .about.58 g) and a
multi-speed adhesion assay (200, 600, 1000, 2000, 2500, and 4000
RPM; .about.6 to 2560 g)..sup.5, 64 Cells were seeded at 10,000
cells/well, then incubated on the surfaces at 4.degree. C. with
total adhesion times of 18 min. After centrifugation, the number of
attached cells was quantified fluorescently with the CyQuant
reagent (Molecular Probes, OR). Plates were read using a Spectramax
Gemini XS fluorescent plate reader (Molecular Devices, CA).
[0090] RCO adhesion strength scaled with ligand density (1-20
pmol/cm.sup.2) and was dependent on ligand type (FIG. 2).
Independent of ligand density, the percent/extent of matrix
mineralization varied with ligand type with a ligand identity trend
similar but not identical to adhesion.
[0091] FIG. 2. Multi-speed adhesion assay results: the detachment
force adhesion parameter, ((F.sub.70).sup.0.5, g.sup.1/2), which
was determined using a reverse normal distribution for each of the
RGD-containing peptides arranged according to ligand with density
in pmol/cm.sup.2. TCPS with adsorbed serum proteins as reference.
Modulation of cell adhesion via ligand type (.psi.) and ligand
density (pmol/cm.sup.2). Note that with a proper choice of ligand
type and ligand density, adhesion greater than tissue culture
polystyrene (TCPS) can be achieved. (within each ligand group
differences are grouped by symbol; p<0.05; Kruskal-Wallis with
Dunn's posthoc test).
[0092] These studies demonstrate the dependence of cell
differentiation (i.e., matrix mineralization for osteoblasts) on
ligand type, ligand density, and adhesion strength. Significantly,
it was demonstrated that short peptides containing the minimal RGD
sequence do not in this case serve as effective ligands for
integrin receptors, whereas the longer counterpart peptides with
more amino acid sequence context do. Furthermore, the high
throughput character of the method enabled the efficient
investigation of multiple ligands at multiple densities and thereby
provided an excellent tool for studying ligand-receptor
interactions under normal cell culture conditions, capabilities
essential for studying analogous interactions for hESCs.
IPN Surfaces Promote the Self-Renewal of Adult Neural Stem
Cells
[0093] Neural stem cells are typically cultured on surfaces
consisting of a poly(styrene)-based material with a passively
adsorbed, animal-derived extracellular matrix (ECM) protein such as
laminin or fibronectin..sup.67, 69 However, both animal- and
human-derived ECM or proteins likely contain variable splice and
glycoforms, offer limited micro- and nano-scale control of
solid-phase signaling, pose problems with lot to lot variability,
and otherwise present problems for therapeutic application..sup.16,
70 The ability of a peptide-modified interpenetrating polymer
network (IPN) synthesized from acrylamide (AAm), poly(ethylene
glycol) monomethyl ether monomethacrylate (pEGMA), and acrylic acid
(AAc) monomers to support NSC growth was explored. NSCs isolated
from the adult rat hippocampus,.sup.67 were seeded onto 15
amino-acid bone sialoprotein RGD peptide [bsp-RGD(15)]-modified
IPNs at various cell densities over four orders of magnitude. This
peptide was chosen for its specificity for
.alpha..sub.v.beta..sub.3 integrin,.sup.64 since NSCs express
.beta..sub.3 integrins and since .alpha..sub.v.beta..sub.3 is known
to engage the ECM molecule laminin..sup.71 Under proliferating
media conditions (20 ng/ml fibroblast growth factor (FGF)-2), cell
adhesion and morphology on the RGD surfaces were similar to that on
laminin (FIG. 3a-b). By contrast, on surfaces with either lower or
no bsp-RGD(15), cells did not adhere effectively (FIG. 3a-d) and
resembled NSC growth in suspension as neurospheres..sup.72 Such
cell aggregates provide less precise control over the cellular
microenvironment, due in part to spatial gradients in signaling and
nutrients and at times internal necrosis. The negative control
bsp-RGE(15) (CGGNGEPRGETYRAY; SEQ ID NO:58), which differs from the
bsp-RGD(15) peptide by only a methylene group, did not support
attachment and thus highlighted the specificity of the NSC
engagement with the peptide-modified IPN. IPNs modified with
bsp-RGD(15) supported NSC proliferation in a ligand dose-dependent
fashion, and IPNs with the highest bsp-RGD(15) density actually
supported slightly faster cell proliferation than standard
laminin-coated surfaces (FIG. 3e, p<0.05).
[0094] FIG. 3.a-d) Bright field images of neural stem cells grown
on peptide-modified IPNs or laminin-I in proliferating media
conditions (1.2 nM FGF-2). e) Growth curves for proliferation of
neural stem cells as assayed by a total nucleic acid stain. Data
represent mean.+-.standard deviation of 3-5 samples. Surfaces not
in the same group (*, .sctn., .dagger., or .dagger-dbl.) were
statistically different from one another (p<0.05; ANOVA between
groups with Tukey-Kramer Honestly Significant Difference post-hoc
test).
[0095] To determine whether the cells were truly undergoing a
process analogous to self-renewal, i.e. proliferation in the
absence of differentiation, quantitative reverse transcription
polymerase chain reaction (qRT-PCR) was employed to quantify the
expression levels of nestin, a marker of an immature neural
cell..sup.73 It was found that nestin expression levels on laminin
and the synthetic hydrogel were the same (FIG. 4).
[0096] Stem cells are defined not only by their self-renewal but
also their ability to undergo differentiation into one or more
phenotypes. FGF-2 was removed and 0.2 .mu.M retinoic acid and 5
.mu.M forskolin, components known to induce tripotent cell
differentiation, were added..sup.69 It was found that cells
differentiated into neurons, astrocytes, and oligodendrocytes, the
three major lineages of the nervous system (FIG. 4). Importantly,
qRT-PCR (and immunostaining) revealed that the expression levels
for markers of these three lineages were statistically
indistinguishable on laminin and the synthetic hydrogel (FIG. 4).
Therefore, the synthetic IPN, displaying an integrin-binding
peptide, was able to substitute for laminin in supporting NSC
proliferation and differentiation.
[0097] The effects of varying peptide identity and density on NSC
differentiation were explored. The peptide motif
Ile-Lys-Val-Ala-Val (IKVAV; SEQ ID NO:59) within laminin has been
shown to promote neurite outgrowth from neurons..sup.74-76 It was
hypothesized that incorporating a 19-mer peptide containing IKVAV
(SEQ ID NO:59) may enhance NSC differentiation into neurons;
however, it was found that as the IKVAV (SEQ ID NO:59) dosage
increased, neuronal differentiation actually decreased (FIG.
4d).
[0098] FIG. 4.a) immunofluorescence staining for the neural stem
cell marker nestin (green) with stained nuclei (blue) in cells
proliferating on laminin or 21 pmol.cm.sup.-2 bsp-RGD(15) modified
hydrogels (media conditions: 1.2 nM FGF-2). qRT-PCR demonstrates
that statistically indistinguishable levels of nestin are expressed
in cells on each surface. Likewise, qRT-PCR indicates that equal
levels of b, the early neuronal marker .beta.-tubulin III, and c,
the mature astrocyte marker glial fibrillary acidic protein (GFAP)
are expressed in cells on either surface under either proliferating
or differentiating media conditions. The box plots summarize the
distribution of points, where the thick line signifies the median
and the ends of the box are the 25th and 75th quartiles. Within
each plot, levels not connected by same letter are significantly
different (p<0.05). d) As the density of bsp-RGD(15) was
decreased and bsp-RGE(15) was increased, neuronal differentiation
decreased. Furthermore, as bsp-RGD(15) was decreased and
lam-IKVAV(19) (SEQ ID NO:59) increased, neuronal marker expression
also decreased.
Example 2
Bacterial Peptide Display and Selection to Identify Novel
Cell-Binding Peptides
[0099] As described above, bacterial display technology is
particularly advantageous, since this approach enables presentation
of pendant peptides on an extended surface (i.e. the bacterial
cell) at densities that can be well-controlled within the range of
1,000-10,000 peptides/bacterial cell..sup.77 To accomplish this, a
novel bacterial display scaffold was developed that enables
identification of peptides that retain their ability to bind to
their target receptor, even when removed from the bacterial surface
scaffold protein used for their isolation. In other words, peptides
discovered by this approach are ideally suited for materials
functionalization through grafting. This new display system enables
presentation of linear or disulfide constrained peptides on the
surface of bacteria as N- or C-terminal fusions to a circularly
permuted variant of outer membrane protein OmpX (CPX), rather than
as insertion fusions. A panel of fluorescent libraries of linear
and constrained peptide libraries displayed as either N- or
C-terminal fusions to CPX was constructed. These libraries are
typically composed of >10.sup.9 independent peptide
sequences/clones. Co-expression of a FACS optimized green
fluorescent protein provides an intrinsic label that enables
library screening using FACS, and streamlined analysis of isolated
clones..sup.78
[0100] FIG. 5. Isolation of Cell-Specific Peptides Using Bacterial
Display. Target cells were incubated with a peptide library
displayed on the surface of bacteria (which are expressing GFP
intracellularly and are therefore fluorescent). The cells bound to
bacteria were thus rendered/labeled fluorescent and were sorted by
FACS, and bound bacteria were recovered by growth for sequence
analysis. Flow cytometric analysis of target cell fluorescence is
shown a) in the absence of bacteria, b) after incubation with the
fluorescent bacterial library, and c) after incubation with an
individual binding clone isolated from the library. Red
auto-fluorescence was also measured to provide improved
discrimination of the target cells in the polygon gate. d) Overlaid
phase contrast and fluorescence image of bacteria bound to a target
cell surface.
[0101] It has been demonstrated that bacterial display methodology
is highly effective in identifying peptides that recognize tumor
cell and erythrocyte surface antigens. In both cases, isolated
peptides: i) were specific for their intended target cell, ii)
could subsequently be chemically synthesized and used to
functionalize materials surfaces (e.g. polystyrene nanoparticles),
and iii) retained binding function even when chemically synthesized
and tested outside of the context of the bacterial display
scaffold. The identification of peptides that recognize both
adherent and suspended cells is straightforward (FIG. 5). Target
cells are mixed together with the library and washed several times,
and the resulting fluorescently-labeled target cells (carrying
bound bacteria) are recovered using FACS. Individual
bacteria/peptide clones are isolated, and plasmid DNA is
recovered/purified for sequencing to determine the identity of the
displayed peptide. This full process can be completed in several
days.
[0102] This technology has also been used to identify peptides
capable of binding to the surface of adult neural stem cells. Both
N- and C-terminal peptide display libraries were screened on NSCs,
with the first two rounds selected through centrifugal pelleting of
NSCs to isolate bacterial clones displaying binding peptides, and
the third round selected through FACS. Following the third round,
numerous peptide clones were sequenced (.about.30 novel sequences
determined to date). Importantly, a number contain RGD motifs,
indicating that the technology likely isolated novel integrin
ligands, whereas others intriguingly do not (FIG. 6a). A BLAST
search did not reveal significant sequence identity to other known
mammalian proteins in most peptides, indicating the identification
of novel cell-binding ligands. Furthermore, to confirm the ability
of the peptides to mediate binding, clonal bacteria populations
presenting each peptide were mixed with NSCs and subjected to flow
cytometry analysis, and results indicate that the displayed
peptides mediate specific binding (FIG. 6b-c).
[0103] FIG. 6. Isolation of Neural Stem Cell Binding Bacterial
Clones. a) Bacterial peptide display libraries were screened as in
FIGS. 5, and 6 representative clones (of .about.30 sequenced to
date) are shown. Three have RGD sequences (number 1 with homology
to thrombospondin and number 3 with homology to collagen IV), and
three do not. b) Individual clonal bacterial populations were
validated for the ability to bind to NSCs. Negative control flow
cytometry data show diagonal fluorescence (with some potential
nonspecific binding not observed in FIG. 5). c) However, all clones
analyzed to date exhibit strong binding to NSCs, as indicated by
the presence of a strongly GFP+ population (relative to their
autofluorescence) of NSCs with adherent bacteria. The
representative data shown are for bacteria presenting peptide 1
from part a).
Example 3
hESCs Grown on Synthetic ECMs
[0104] The work with osteoprogenitors (FIG. 2) and neural stem
cells (FIGS. 3-4) establishes the strong potential of IPN surfaces
to control cell function.
[0105] The HSF6 hESC cell line, a federally approved line derived
at UCSF.sup.80, was used. This line was cultured on both mouse
embryonic fibroblast (MEF) feeder cells as well as a
semi-interpenetrating network hydrogel system. MEFs (from CF-1
mice, Charles River) were mitotically inactivated via gamma
irradiation and cultured on gelatin (collagen-derived) adsorbed to
plasma-treated polystyrene (Falcon). Complete culture media (KSR)
consisted of: Knockout-DMEM (Invitrogen), 20% Knockout Serum
Replacement (Invitrogen), 2 mM Glutamine (Invitrogen), 0.1 mM
non-essential amino acids (NEAA) (Invitrogen), 0.1 mM
.beta.-mercaptoethanol (Sigma), and 4 ng/mL FGF-2 (R&D
Systems). When hESCs were cultured on sIPNs, colonies were
maintained in conditioned KSR media, generated by pre-incubating
KSR on MEFs for 24 hours such that the hESCs could be exposed to
signaling molecules secreted from MEFs.
[0106] To compare the ability of sIPNs vs. MEFs to support the
self-renewal of hESCs, several characteristics were assessed:
colony attachment, colony morphology, cell viability, and the
presence of hESC markers. Morphological changes were one of the
early indicators of differentiation. Undifferentiated hESC colonies
that were cultured on MEFs (positive control) are shown in FIG.
7a-b. Undifferentiated hESCs exhibited a high nucleus to cytoplasm
ratio, formed tightly packed colonies with defined colony borders,
and expressed embryonic stem cell markers such as the transcription
factor Oct4 and the surface carbohydrate moieties stage-specific
embryonic antigen SSEA-3 and SSEA-4 (not shown). FIG. 7e-f shows
that very few hESC colonies were able to attach to a negative
control gelatin-coated surface. Also, the few that attached
spontaneously differentiated, resulting in indistinct colony
borders and larger, spindly, fibroblast-like cells that migrated
away from the colony. In contrast, hESCs cultured on the sIPNs
(FIG. 7c-d) exhibited morphologies similar to those of
undifferentiated hESCs cultured on MEFs (FIG. 7a-b), where colonies
had distinct borders with small (.about.10 .mu.M diameter) and
tightly packed cells.
[0107] FIG. 7. Morphology and Oct4 immunofluorescence of hESCs at
Day 5. a, b) hESCs cultured on MEFs exhibited small, tightly packed
cells with distinct colony borders. c, d) hESCs cultured on sIPN
exhibited similar morphologies when compared to a, b. e, f) hESCs
cultured on gelatin-adsorbed polystyrene exhibited morphologies of
spontaneously differentiating cells, with spindle-shaped cells and
indistinct colony borders. Oct4 was present in some cells under all
three conditions. However, note that in hESCs cultured on
polystyrene (1), white arrows point to cells beyond the colony edge
which were not positive for Oct4.
[0108] Immunofluorescence staining was conducted to assess whether
cells retained markers of undifferentiated hESCs. The POU family
transcription factor Oct4 is a highly specific marker, and
necessary protein, for undifferentiated hESCs, and SSEA-4 is a
glycolipid cell surface antigen strongly expressed in
undifferentiated hESCs..sup.18 Results showed the presence of Oct4
(FIG. 7) and SSEA-4 in cultures of all three conditions at day 5.
For the hESCs cultured on gelatin-adsorbed polystyrene (FIG. 7f),
cells beyond the edge of the colony were not positive for Oct4,
indicating that they had spontaneously differentiated. Under these
suboptimal conditions, some hESCs in the colony cores did not yet
completely lose their undifferentiated characteristics after 5
days..sup.81 By comparison, the hESCs cultured on sIPNs (FIG. 7d)
exhibited a tight border and were positive for Oct4. Interestingly,
the Oct4 fluorescence appeared somewhat diffuse in the center of
the colony, a result attributed to competing fluorescence from
out-of-focus cell layers in the colony.
[0109] To take an important step towards a fully chemically defined
microenvironment, H1 hES cells were cultured,.sup.17 for which a
serum-free, defined medium has been developed. In addition, this
feeder-free system involves culturing cells on tissue culture
polystyrene coated with Matrigel, an ECM mixture of predominantly
fibronectin, collagen IV, and heparan sulfate..sup.19, 82 Cells
were grown in non-conditioned, serum free medium (NC-SFM), which
consists of X-VIVO 10 (Cambrex, Walkersville, Md.) medium
supplemented with 0.5 ng/mL TGF-.beta.1 and 80 ng/mL FGF-2
(Invitrogen). This medium and substrate, developed by Geron Corp.,
have been found to support hESC self-renewal to the same to the
same extent as MEF feeders..sup.82 Cells are passaged by mechanical
disruption, most often after brief incubation in collagenase
IV.
[0110] Cultures on Matrigel consisted of Oct4.sup.+ hESC colonies
interspersed with differentiated, Oct4-stromal cells derived from
the hESCs, as previously described (note that the hESCs can be
selectively passaged by timing the enzymatic digest)..sup.82 H1
cells were also cultured in the NC-SFM on bsp-RGD(15) conjugated
IPN surfaces (identical to those in FIGS. 3-4). Cultures were
strikingly similar to those cultured on Matrigel, with high Oct4
levels in large hESC colonies. By contrast, cells predominantly
died on bsp-RGE(15) IPN surfaces and differentiated on polystyrene.
Both HSF6 cells on the sIPN (FIG. 7) and H1 cells on the IPN were
able to maintain Oct4 expression for 3 passages (.about.10
days).
Example 4
Screening Peptide Libraries for Activation of Cell Signaling
Pathways
[0111] In general, soluble growth factor signals synergistically
interact with immobilized matrix signals to regulate cell
function..sup.88, 89 However, even if an immobilized motif binds to
a cell surface, it may not actively stimulate cellular signaling
pathways. Therefore, while the peptides isolated as described above
likely contain a rich repertoire of binding motifs for tissue
engineering applications, it would be highly advantageous to select
them for bioactivity.
[0112] The phosphoinositide-3 kinase (PI 3-kinase) signaling
pathway, which can be activated by numerous cell surface receptors,
has been implicated in regulating cell survival and proliferation
in a number of contexts..sup.90-92 After ligation and activation of
canonical receptors, phosphorylation of receptor tails on specific
tyrosine residues recruits PI 3-kinase to the cell surface to
phosphorylate phosphoinositide lipids. These lipids then recruit
several kinases, including PDK1 and Akt. Phosphorylation of the
latter by the former activates Akt, which then modulates a number
of downstream effectors (such as Bad, mTOR, forkhead transcription
factors, GSK-3.beta., and others) important in cell survival,
proliferation, and other functions..sup.92, 93 Very importantly, PI
3-kinase and Akt signaling may be a general pathway important for
stem cell proliferation and self-renewal. It has previously been
found that their signaling (but not MAPK signaling) is required for
self-renewal, as PI3-kinase/Akt inhibition leads to ES
differentiation..sup.94, 95 Likewise, it has been found that PI
3-kinase and Akt signaling is activated by numerous adult neural
stem cell mitogens (FIG. 8a, c-g). Furthermore, growing neural stem
cells on the biomimetic hydrogel displaying bsp-RGD(15) led to the
activation of Akt to a greater extent than a surface displaying the
negative control bsp-RGE(15) peptide (the same surfaces as in FIGS.
3-4). Finally, chemical or genetic inhibition of this pathway
inhibits self-renewal, whereas overexpression of transducers in
this pathway promotes self-renewal in a mitogen-independent
fashion.
[0113] Fluorescence resonance energy transfer has served as the
basis for high throughput drug screens,.sup.96 and can be used to
screen peptides. A FRET sensor of Akt activation has already been
developed. Briefly, a donor cyan fluorescent protein (CFP) and
acceptor yellow fluorescent protein (YFP) with an intervening Akt
substrate peptide yields FRET signal changes with Akt signaling and
phosphorylation..sup.6 This sensor is compatible with a high
throughput, FACS-based screen.
[0114] Experimental Design: CFP and YFP mutants that are optimal
FRET partners,.sup.97 were recently identified in a review article
as the optimal FRET partners in the field..sup.98 To further
enhance the sensitivity of the existing Akt FRET sensor, the more
sensitive CFP/YFP pair is used..sup.97 A hESC cell line stably
expressing this enhanced Akt sensor is generated using a lentiviral
vector system.sup.99, 100 that has previously proven to be an
effective vector for generating stable hESC cell lines..sup.101,
102
[0115] The genes encoding the scaffold protein and successful
binding peptides from are amplified by PCR, inserted into an
otherwise identical expression plasmid that does not contain the
GFP (which would interfere with FRET), and transformed into
bacteria. The resulting bacterial library is incubated with the
hESC line in suspension as described above, and after 5-30 minutes
(as in FIG. 8e-f), cells are subjected to flow cytometry to screen
for peptide clones capable of activating PI 3-kinase signaling in
hESCs and thereby inducing a FRET signal. Additional such selection
rounds may be conducted if necessary. This FRET-based FACS screen
will cull the peptides to yield a smaller number of peptides
capable of activating hESC signal transduction.
[0116] FIG. 8. Western blotting shows the level of
phosphorylated/activated Akt (top rows) vs. total Akt (bottom rows)
after a) stimulation with FGF-2 (minutes). b) Furthermore, higher
levels of Akt are activated on RGD vs. RGE hydrogels. c) and d)
Immunostaining for phospho-Akt shows Akt activation by FGF-2.
Likewise relocalization of a PH-GFP genetic sensor to the cell
surface acts as a sensor of PI 3-kinase signal activation after
FGF-2 addition. An analogous genetic sensor of Akt based on FRET
has been generated..sup.6
[0117] FIG. 9 is a schematic depiction of a Protein Kinase B (PKB)
sensor (BKAR). Incorporation of selected peptides into a synthetic
hydrogel can yield bioactive materials that efficiently promote
long-term hES self-renewal.
[0118] About 20 chemically synthesized peptides (EZBiolabs, 10 mg
synthesis scale, >90% purity) identified above are used, but
with terminal Cys residues for attachment to the matrix..sup.64 The
experimental system described above (FIGS. 1-4) is used to create
IPNs on standard 48-well plates and subsequently graft these
chemically synthesized peptides at various densities. Effective
peptide densities for this work can range from 0 to 20
pmol/cm.sup.2, showing myriad cell behavior such as cell
attachment, spreading, focal contact formation, and proliferation
requires ligand densities on solid 2D and hydrogel surfaces greater
than approximately 1.5 fmol/cm.sup.2 to 10 pmol/cm.sup.2..sup.1,
28, 28, 35, 38, 107, 108 These surface densities translate into
.about.1-100 nmol/cm.sup.3, assuming a 10 nm thick slab of
hydrogel..sup.107
[0119] The centrifuge-based adhesion assay, which employed
peptide-modified IPNs (FIG. 3), is used as a first test of the
adhesive character of these peptides. Importantly, both cell
adhesion strength and intracellular signaling have been observed to
be directly proportional to the number of integrin-ligand
bonds,.sup.109, 110 Since the adhesion assay was developed to test
initial adhesion events, assay times are kept short (<20 min),
and experiments are conducted at 4.degree. C., a standard
experimental practice..sup.111 After seeding 10,000 cells/well from
a 4.degree. C. cell stock, each well is overfilled with 4.degree.
C. media. Overfilling ensures that any bubbles present at this
stage can carefully be removed (aspirated), and results in each
well being topped off with a 1-2 mm positive meniscus to ensure
that air pockets will not be trapped in the wells after subsequent
sealing (Titer-Tops adhesive film, Diversified Biotech, Inc).
Assays are performed using an Eppendorf 5810R centrifuge equipped
with a swinging bucket rotor fitted with microplate buckets
(A-4-62-MTP) (effective radius 14.3 cm; possible detachment forces
6 to 2558 g). To prevent leakage, each swinging bucket is modified
with a stainless steel support plate to replace the stock
semi-rigid plastic supports and provide a flat, rigid surface for
the inverted sample plates to press against without distortion. In
addition, buckets are modified with a .apprxeq.2 mm thick membrane
made of silicone elastomer positioned between the support plate and
the inverted, taped microplate to further prevent well leakage.
[0120] Due to the short assay times required (<20 min), cells
are first forced to the bottom of the wells to engage ligands by
gently pre-spinning plates at a low force (5 min, 4.degree. C.,
.about.6 g). Pre-spinning forces less than 10 g do not
significantly affect the subsequent force required to detach
cells..sup.111 After pre-spinning, plates are incubated at
4.degree. C. for 5 min in the centrifuge (total adhesion time, 18
min). Plates are then inverted, centered on top of the silicone
elastomer and subjected to a .about.57 g detachment force (600
RPM). Following cell detachment, the tape is quickly removed, and
each well is gently aspirated. To prevent detached cells from
resettling, plates remain inverted until the tape is removed. After
aspiration, plates are frozen (-80.degree. C.), and the total
number of attached cells is quantified using CyQuant (FIG. 3).
Running the assay without any rinsing steps to remove non-adherent
cells eliminates the potential of well-to-well and plate-to-plate
variation caused by uncontrolled hydrodynamic forces during
rinses..sup.112
Example 5
Screening Peptide Libraries for Neural Stem Cell Binding
[0121] Three different, validated peptide libraries were pooled to
generate a large library (>10.sup.10 independent clones) and
were used for selection and screening..sup.79 These libraries
include a random 15-mer (--X.sub.15--) fused to either the N- or
C-terminus of the CPX display scaffold and an N-terminal
constrained library of the form X.sub.2CX.sub.7CX.sub.2. The
library has been made intrinsically fluorescent using a bicistronic
expression vector to enable co-expression of a green fluorescent
protein optimized for FACS.
[0122] The bacterial library was screened for binding peptides. For
the first two cycles, screening was again conducted by
co-sedimentation, where as many as 10.sup.8 NSCs suspended via
mechanical disruption in 0.5 mM EDTA as described.sup.82 were
incubated with 10.sup.10 bacteria prior to several gentle
centrifugations and rinses. The resulting mixture was added
directly to bacterial media (LB) for expansion of the binding
clones. The second cycle repeated this process with 10-fold fewer
bacteria for higher stringency. In the third cycle, after
incubation with 10.sup.6 NSCs and washing, cells were sorted (as in
FIGS. 5-6) using a DAKO-Cytomation MoFlo flow cytometer in the UC
Berkeley Cancer Center to stringently isolate bacteria that are
bound to NSCs. The resulting sorted bacteria were expanded. Plasmid
DNA was isolated from a number of individual clones (at least
100).sup.79 and subjected to DNA sequencing to identify the peptide
responsible for binding to the NSC surface.
[0123] Bacterial peptide display libraries were constructed, as
described above, and tested for binding capacity to neural stem
cells. Clonal populations of the bacterial peptide display
libraries after the third round of selection were analyzed via flow
cytometry to measure the binding capacity of the clones. Sequences
of the clones were determined through DNA sequence of the plasmid
expressed by the bacteria. Table 5 presents amino acid sequences of
peptides and the binding capacity to NSCs of bacterial clones
displaying the peptides.
TABLE-US-00005 TABLE 5 % NSCs with Clone Bacteria Peptide Sequence
15-2 75.9 DHKFGLVMLNKYAYAG (SEQ ID NO: 60) Co-3 75.7 GGCRWYAKWVCVW
(SEQ ID NO: 61) Co-9 75.7 SKCWGWTPYYCVA (SEQ ID NO: 62) Co-22 75.6
VWCGMFGKRRCVT (SEQ ID NO: 63) 7C-21 75.5 WNCIKGSSWACVW (SEQ ID NO:
64) 7C-1 75.2 WYCFREN KYVCVM (SEQ ID NO: 65) Co-2 74.8
WSCPKVNQYACFW (SEQ ID NO: 66) Co-12 74.3 WVCLWRHRGDCSI (SEQ ID NO:
67) Co-1 73.8 SLCAAYNRWACIW (SEQ ID NO: 68) Co-10 72.7
WRCLGDGYHACVR (SEQ ID NO: 69) Co-11 72.7 LECPGESKYYCIY (SEQ ID NO:
70) Co-17 72.1 WECAEESKFWCVF (SEQ ID NO: 71) 7C-3 71.9
WFCLLGRSAYCVR (SEQ ID NO: 72) Co-16 71.9 QGCAFVTYWACIF (SEQ ID NO:
73) 7C-9 71.3 KLCCFDKGYYCMR (SEQ ID NO: 74) Co-18 71.3
WWCKKPEYWYCIW (SEQ ID NO: 75) Co-23 71.1 LVCNRQNPWVCYI (SEQ ID NO:
76) Co-15 70.9 WVCNDLIHHFCVW (SEQ ID NO: 77) Co-20 70.9
RLCCWKTQYFCEI (SEQ ID NO: 78) Co-21 70.9 MYCERDSKYWCIH (SEQ ID NO:
79) 15-32 70.4 RRELVRMTDWVWVSG (SEQ ID NO: 80) 15-6 70.2
LEDAMGWALSWGHIW (SEQ ID NO: 81) 7C-8 68.8 WLCLDKNCMACVW (SEQ ID NO:
82) 7C-17 67.8 WLCKGSNKYMCEW (SEQ ID NO: 83) Co-5 67.4
WDCGKKNAWMCIW (SEQ ID NO: 84) 7C-20 65.7 WVCIWERFKSCNE (SEQ ID NO:
85) 7C-5 65.6 IWCGSRFGCWCKP (SEQ ID NO: 86) 15-50 64.9
GFVLVWSYTCRCWGK (SEQ ID NO: 87) Co-13 64.8 STCSWVSSYVCIM (SEQ ID
NO: 88) 7C-12 64.5 FWCIRGEYWVCDR (SEQ ID NO: 89) 15-16 63.9
SDWSVLLSCERWYCI (SEQ ID NO: 90) 15-52 63.3 ESGLKVMCMKYYCMA (SEQ ID
NO: 91) 7C-4 62.0 YMCMSRGDATCDV (SEQ ID NO: 92) 7C-6 61.8
GECFYYVMNTCVW (SEQ ID NO: 93) 7C-24 61.7 WWCDMRGDSRCSG (SEQ ID NO:
94) Co-8 61.7 WTWESAFAGRWEVGD (SEQ ID NO: 95) 7C-2 55.2
ESCWYQIMYKCAN (SEQ ID NO: 96) 7C-14 54.5 LNCAMYNACIW (SEQ ID NO:
97) 7C-15 54.0 QCCQLRGDAVCNC (SEQ ID NO: 98) Co-19 50.6
WQCGRFWCIHCLW (SEQ ID NO: 99) 7C-7 47.8 LECTERGDFNCFV (SEQ ID NO:
100) 7C-22 46.9 WMCSGVQPNACVW (SEQ ID NO: 101) 7C-11 37.7
LCCESYICALCHY (SEQ ID NO: 102) 15-59 22.5 DLCTYGHLWLGNGRP (SEQ ID
NO: 103) 7C-19 20.5 WVCNKLGVYACEY (SEQ ID NO: 55)
[0124] The GFP-expressing bacterial peptide libraries from each
round were incubated with the neural stem cells and the amount of
bacteria bound to these cells was quantified via flow cytometry. A
bacterial population only expressing the membrane protein CPX with
no peptide was also tested in a similar manner. The data, shown in
FIG. 10, indicate that after the second and third rounds of
selection, the library population has a higher amount of bacteria
binding to the neural stem cells compared to binding of bacteria
with no peptide displayed.
Example 6
Screening Peptide Libraries for Embryonic Stem Cell Binding
[0125] The approach described in Example 5, above, is applied to
hESCs. To provide an approach that yields peptides potentially
suitable for hESCs cultured in both serum containing and serum free
media, hES cell lines HSF6 (derived at UCSF, FIG. 7) and H1
(originally derived by Thomson et al..sup.17) can be used.
[0126] In addition, throughout this work, benchmark control cell
culture conditions consisted of culturing cells on surfaces coated
with a 1:30 dilution of Matrigel (growth factor reduced, Becton
Dickinson) in defined, non-conditioned, serum free medium (NC-SFM),
as described..sup.82
[0127] The bacterial library is screened for binding peptides. For
the first two cycles, screening is again conducted by
co-sedimentation, where as many as 10.sup.8 hESCs suspended via
mechanical disruption in 0.5 mM EDTA as described.sup.82 are
incubated with 10.sup.10 bacteria prior to several gentle
centrifugations and rinses. The resulting mixture is added directly
to bacterial media (LB) for expansion of the binding clones. The
second cycle repeats this process with 10-fold fewer bacteria for
higher stringency. In the third cycle, after incubation with
10.sup.6 hESCs and washing, cells are sorted (as in FIGS. 5-6)
using a DAKO-Cytomation MoFlo flow cytometer in the UC Berkeley
Cancer Center to stringently isolate bacteria that are bound to
hESCs. In addition, if needed, cells are sorted that both have
bound, GFP+ bacteria and have high levels of expression of the hESC
marker SSEA-4 (by using a primary antibody against this antigen
followed by a Cy3-labeled secondary antibody,.sup.82 prior to
incubation with bacteria). The resulting sorted bacteria are
expanded. Plasmid DNA is isolated from a number of individual
clones (at least 100).sup.79 and subjected to DNA sequencing to
identify the peptide responsible for binding to the hESC
surface.
Example 7
Biomaterial Screening Through Analysis of NSC Attachment and
Proliferation
[0128] Peptides containing a FITC molecule were grafted onto IPN
surfaces at various peptide concentrations. The amount of peptide
bound to the surface was quantified by cleaving off the FITC tag
with chymotrypsin and measuring the resulting fluorescence. The
results are shown in FIG. 11. The 7C-9 peptide was found in the
bacterial peptide display selections and is a novel peptide. The
RGD peptide has been shown to allow for similar cell attachment and
proliferation on the IPN surface in comparison to laminin-coated
surfaces. The 7C-9 peptide attaches to the surface at lower
concentrations in comparison to the RGD peptide though it can
saturate the surface at high peptide grafting concentration.
[0129] IPNs were grafted with RGD, 7C-9, and 7C-24 peptides at
various concentrations. After allowing neural stem cells attach to
the surface for 6 hours, the amount of cells was quantified with
Cyquant, a fluorescent DNA-binding dye. The data are shown in FIG.
12. The cell attachment was similar for all surfaces except for RGE
where the amount of cells was below the detection limit of the
assay. *=Below detection limit of the assay.
[0130] Cell proliferation was quantified on peptide-grafted IPN
surfaces after 5 days with Cyquant. The cell number was normalized
to the amount of cells seeded on the IPN surfaces. The data are
shown in FIG. 13. The RGD-grafted surfaces show a much higher
amount of cell proliferation than 7C-9 and 7C-24-grafted surfaces
though 7C-9 surfaces have a lower peptide surface concentration
compared to the RGD surfaces. A control surface with an RGE peptide
was shown to have little or no cells detached. *=Below the
detection limit of the assay.
[0131] Cell proliferation was quantified on peptide-grafted IPN
surfaces after 5 days with Cyquant. The cell number was normalized
to the amount of cells seeded on the IPN surfaces. The data are
shown in FIG. 14. Comparing the 7C-9 peptide surfaces at low
peptide surface concentrations with the RGD surfaces at similar
peptide concentrations, it appears that there is slower
proliferation on the 7C-9 surfaces.
Example 8
Biomaterial Screening Through Analysis of Stem Cell Function and
Self-Renewal
[0132] For a smaller set of as many as 6 peptides that support
attachment, the following comprehensive assays are conducted in
vitro and in vivo to analyze hESC behavior on the hydrogels: 1)
cell viability, 2) relative Akt signal activation, 3) cell
proliferation, 4) SSEA-4 and Tra-1-80 self-renewal marker analysis,
5) Oct4 self-renewal marker analysis, 6) hTERT self-renewal marker
analysis, 7) karyotype analysis, 8) embryoid body differentiation,
and 9) teratoma formation. In addition, the following control
surfaces are used: 1) tissue culture polystyrene with adsorbed
gelatin (negative control for self-renewal, as in FIG. 7), 2) MEFs
(positive control for self-renewal, as in FIG. 7), 3) Matrigel
(positive control),.sup.82 4) sIPNs with no peptide (negative cell
attachment control), 5) sIPNs with the bsp-RGE(15) peptide
(negative cell attachment control, as in FIG. 8), and 6) sIPNs with
the bsp-RGD(15) peptide (benchmark control, as in FIG. 8).
[0133] Cell viability (assayed after 48 hours) is examined using
the calcein-AM stain (Molecular
[0134] Probes) and plotted as a function of peptide concentration.
Steady state (48 hour) Akt activation is assayed by Western
blotting (FIG. 8). Proliferation data (assayed after 96 hours) are
collected using the CyQuant assay (FIG. 3) and likewise plotted as
a function of peptide concentration.
[0135] More detailed, long-term biological analysis is conducted on
as many as 6 hydrogel surfaces (i.e. peptides at a specific
concentration) that yield the most robust viability, signaling, and
proliferation responses. For these medium-term studies, the
peptides are incorporated into the same IPN hydrogel demonstrated
to have promise for hESC culture. hESC maintenance in an
undifferentiated state after five cell passages on the surfaces is
assessed via numerous standard approaches established in the
literature.sup.82, 87, 113. Morphology and cell/nucleus ratios are
assessed qualitatively, as demonstrated in FIG. 7. Immunostaining
is combined with flow cytometry to quantify the percentage of cells
expressing key hESC markers..sup.82 Briefly, cells are dissociated
using a 0.5 mM EDTA solution, blocked with rabbit serum, incubated
in primary antibodies for SSEA-4 and Tra-1-80 (Chemicon), and
incubated with the appropriate Alexa 488, Cy3, or Cy5-labeled
secondary rabbit antibodies (Jackson ImmunoResearch). Cells are
then analyzed via flow cytometry. Taqman QPCR is conducted
essentially as above (FIG. 5, Preliminary Studies) using previously
developed primers and probes to quantify hESC transcription factor
expression (Oct4) and telomerase activity (hTERT: the catalytic
subunit of telomerase)..sup.82 To move towards fully defined
systems, studies are conducted with H1 cells.sup.17 (in NC-SFM
medium) and are confirmed with HSF6 cells (in KSR medium).
[0136] Cells are also cultured on IPNs coated with bsp-RGD(15) in
NC-SFM plus all chemically synthesized soluble peptides at 100 nM
and 1 .mu.M for five passages. If colonies morphologically
consistent with hESC colonies are still present (FIG. 7), cells are
subjected to SSEA-4 immunostaining and Oct4 QPCr. Note that Oct4+
hESCs could only be successfully expanded on bsp-RGD(15) for three
passages.
[0137] For sIPNs that maintain hESC markers after five passages, a
parallel experiment is conducted for 10 passages and self-renewal
re-assessed as defined above. Matrices that support self-renewal at
10 passages are used to analyze chromosome stability and the
ability of the cells to undergo differentiation. Specifically,
cytogenetic analysis of 20-50 cells is performed using GTG-banding
at the Medical Genetics Cytogenetics Laboratory (Children's
Hospital, Oakland, Calif.), as previously described..sup.82
Furthermore, hESCs maintained in an immature state retain the
ability to undergo differentiation into cells of the three germ
layers. To assess the in vitro differentiation capacity of
cultures, embryoid body differentiation analysis is conducted by
collagenase dissociation of cultures after 10 passages into small
clumps, followed by suspension in low attachment culture plates
(Corning). Differentiation is conducted in 80% KO-DMEM, 20% FBS,
and supplements as described.sup.82. After 4 days in suspension
followed by 10 days on chamber slides, cells are processed and
stained for ecotodermal (.beta.-tubulin III staining for neurons,
Sigma antibody), mesodermal (muscle actin staining, Dako Corp.
antibody), and endodermal (.alpha.-fetoprotein, Sigma antibody)
lineages. Finally, to assay the in vivo differentiation
capabilities of hESCs expanded on synthetic surfaces, teratomas are
formed via intramuscular injection of 5 million cells into the
hindlimb of SCID/beige mice. After 75 days, tissue is analyzed by
IDEXX for the presence of differentiated cells from the three germ
layers (West Sacramento, Calif.), as described..sup.15, 17, 19,
82
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[0283] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
103111PRTArtificial SequenceConsensus sequence 1Arg Lys Arg Asp Arg
Leu Gly Thr Leu Gly Ile1 5 10 215PRTArtificial SequenceSynthetic
Peptide 2Cys Gly Gly Asn Gly Glu Pro Arg Gly Asp Thr Tyr Arg Ala
Tyr1 5 10 15 312PRTArtificial SequenceSynthetic Peptide 3Cys Glu
Pro Arg Gly Asp Thr Tyr Arg Ala Tyr Gly1 5 10 415PRTArtificial
SequenceSynthetic Peptide 4Val Ser Trp Phe Ser Arg His Arg Tyr Ser
Pro Phe Ala Val Ser1 5 10 15 511PRTArtificial SequenceSynthetic
Peptide 5Cys Thr Arg Lys Lys His Asp Asn Ala Gln Cys1 5 10
621PRTArtificial SequenceSynthetic Peptide 6Lys Gln Asn Cys Leu Ser
Ser Arg Ala Ser Arg Gly Cys Val Arg Asn1 5 10 15 Leu Arg Leu Ser
Arg 20 716PRTArtificial SequenceSynthetic Peptide 7Asp His Lys Phe
Gly Leu Val Met Leu Asn Lys Tyr Ala Tyr Ala Gly1 5 10 15
815PRTArtificial SequenceSynthetic Peptide 8Leu Glu Asp Ala Met Gly
Trp Ala Leu Ser Trp Gly His Ile Trp1 5 10 15 915PRTArtificial
SequenceSynthetic Peptide 9Ser Asp Trp Ser Val Leu Leu Ser Cys Glu
Arg Trp Tyr Cys Ile1 5 10 15 1015PRTArtificial SequenceSynthetic
Peptide 10Arg Arg Glu Leu Val Arg Met Thr Asp Trp Val Trp Val Ser
Gly1 5 10 15 1115PRTArtificial SequenceSynthetic Peptide 11Gly Phe
Val Leu Val Trp Ser Tyr Thr Cys Arg Cys Trp Gly Lys1 5 10 15
1215PRTArtificial SequenceSynthetic Peptide 12Glu Ser Gly Leu Lys
Val Met Cys Met Lys Tyr Tyr Cys Met Ala1 5 10 15 1315PRTArtificial
SequenceSynthetic Peptide 13Asp Leu Cys Thr Tyr Gly His Leu Trp Leu
Gly Asn Gly Arg Pro1 5 10 15 1413PRTArtificial SequenceSynthetic
Peptide 14Trp Tyr Cys Phe Arg Glu Asn Lys Tyr Val Cys Val Met1 5 10
1513PRTArtificial SequenceSynthetic Peptide 15Glu Ser Cys Trp Tyr
Gln Ile Met Tyr Lys Cys Ala Asn1 5 10 1613PRTArtificial
SequenceSynthetic Peptide 16Trp Phe Cys Leu Leu Gly Arg Ser Ala Tyr
Cys Val Arg1 5 10 1713PRTArtificial SequenceSynthetic Peptide 17Tyr
Met Cys Met Ser Arg Gly Asp Ala Thr Cys Asp Val1 5 10
1813PRTArtificial SequenceSynthetic Peptide 18Ile Trp Cys Gly Ser
Arg Phe Gly Cys Trp Cys Lys Pro1 5 10 1913PRTArtificial
SequenceSynthetic Peptide 19Gly Glu Cys Phe Tyr Tyr Val Met Asn Thr
Cys Val Trp1 5 10 2013PRTArtificial SequenceSynthetic Peptide 20Leu
Glu Cys Thr Glu Arg Gly Asp Phe Asn Cys Phe Val1 5 10
2113PRTArtificial SequenceSynthetic Peptide 21Trp Leu Cys Leu Asp
Lys Asn Cys Met Ala Cys Val Trp1 5 10 2213PRTArtificial
SequenceSynthetic Peptide 22Lys Leu Cys Cys Phe Asp Lys Gly Tyr Tyr
Cys Met Arg1 5 10 2313PRTArtificial SequenceSynthetic Peptide 23Leu
Cys Cys Glu Ser Tyr Ile Cys Ala Leu Cys His Tyr1 5 10
2413PRTArtificial SequenceSynthetic Peptide 24Phe Trp Cys Ile Arg
Gly Glu Tyr Trp Val Cys Asp Arg1 5 10 2511PRTArtificial
SequenceSynthetic Peptide 25Leu Asn Cys Ala Met Tyr Asn Ala Cys Ile
Trp1 5 10 2613PRTArtificial SequenceSynthetic Peptide 26Gln Cys Cys
Gln Leu Arg Gly Asp Ala Val Cys Asn Cys1 5 10 2713PRTArtificial
SequenceSynthetic Peptide 27Trp Leu Cys Lys Gly Ser Asn Lys Tyr Met
Cys Glu Trp1 5 10 2813PRTArtificial SequenceSynthetic Peptide 28Trp
Val Cys Asn Lys Leu Gly Val Tyr Ala Cys Glu Tyr1 5 10
2913PRTArtificial SequenceSynthetic Peptide 29Trp Val Cys Ile Trp
Glu Arg Phe Lys Ser Cys Asn Glu1 5 10 3013PRTArtificial
SequenceSynthetic Peptide 30Trp Asn Cys Ile Lys Gly Ser Ser Trp Ala
Cys Val Trp1 5 10 3113PRTArtificial SequenceSynthetic Peptide 31Trp
Met Cys Ser Gly Val Gln Pro Asn Ala Cys Val Trp1 5 10
3213PRTArtificial SequenceSynthetic Peptide 32Trp Trp Cys Asp Met
Arg Gly Asp Ser Arg Cys Ser Gly1 5 10 3313PRTArtificial
SequenceSynthetic Peptide 33Ser Leu Cys Ala Ala Tyr Asn Arg Trp Ala
Cys Ile Trp1 5 10 3413PRTArtificial SequenceSynthetic Peptide 34Trp
Ser Cys Pro Lys Val Asn Gln Tyr Ala Cys Phe Trp1 5 10
3513PRTArtificial SequenceSynthetic Peptide 35Gly Gly Cys Arg Trp
Tyr Ala Lys Trp Val Cys Val Trp1 5 10 3613PRTArtificial
SequenceSynthetic Peptide 36Trp Asp Cys Gly Lys Lys Asn Ala Trp Met
Cys Ile Trp1 5 10 3715PRTArtificial SequenceSynthetic Peptide 37Trp
Thr Trp Glu Ser Ala Phe Ala Gly Arg Trp Glu Val Gly Asp1 5 10 15
3813PRTArtificial SequenceSynthetic Peptide 38Ser Lys Cys Trp Gly
Trp Thr Pro Tyr Tyr Cys Val Ala1 5 10 3913PRTArtificial
SequenceSynthetic Peptide 39Trp Arg Cys Leu Gly Asp Gly Tyr His Ala
Cys Val Arg1 5 10 4013PRTArtificial SequenceSynthetic Peptide 40Leu
Glu Cys Pro Gly Glu Ser Lys Tyr Tyr Cys Ile Tyr1 5 10
4113PRTArtificial SequenceSynthetic Peptide 41Trp Val Cys Leu Trp
Arg His Arg Gly Asp Cys Ser Ile1 5 10 4213PRTArtificial
SequenceSynthetic Peptide 42Ser Thr Cys Ser Trp Val Ser Ser Tyr Val
Cys Ile Met1 5 10 4313PRTArtificial SequenceSynthetic Peptide 43Trp
Val Cys Asn Asp Leu Ile His His Phe Cys Val Trp1 5 10
4413PRTArtificial SequenceSynthetic Peptide 44Gln Gly Cys Ala Phe
Val Thr Tyr Trp Ala Cys Ile Phe1 5 10 4513PRTArtificial
SequenceSynthetic Peptide 45Trp Glu Cys Ala Glu Glu Ser Lys Phe Trp
Cys Val Phe1 5 10 4613PRTArtificial SequenceSynthetic Peptide 46Trp
Trp Cys Lys Lys Pro Glu Tyr Trp Tyr Cys Ile Trp1 5 10
4713PRTArtificial SequenceSynthetic Peptide 47Trp Gln Cys Gly Arg
Phe Trp Cys Ile His Cys Leu Trp1 5 10 4813PRTArtificial
SequenceSynthetic Peptide 48Arg Leu Cys Cys Trp Lys Thr Gln Tyr Phe
Cys Glu Ile1 5 10 4913PRTArtificial SequenceSynthetic Peptide 49Met
Tyr Cys Glu Arg Asp Ser Lys Tyr Trp Cys Ile His1 5 10
5013PRTArtificial SequenceSynthetic Peptide 50Val Trp Cys Gly Met
Phe Gly Lys Arg Arg Cys Val Thr1 5 10 5113PRTArtificial
SequenceSynthetic Peptide 51Leu Val Cys Asn Arg Gln Asn Pro Trp Val
Cys Tyr Ile1 5 10 5212PRTArtificial SequenceSynthetic Peptide 52Cys
Gly Gly Glu Pro Arg Gly Asp Thr Tyr Arg Ala1 5 10 538PRTArtificial
SequenceSynthetic Peptide 53Cys Gly Pro Arg Gly Asp Thr Tyr1 5
549PRTArtificial SequenceSynthetic Peptide 54Cys Gly Pro Arg Gly
Asp Thr Tyr Gly1 5 5513PRTArtificial SequenceSynthetic Peptide
55Trp Val Cys Asn Lys Leu Gly Val Tyr Ala Cys Glu Tyr1 5 10
5610PRTArtificial SequenceSynthetic Peptide 56Cys Gly Gly Phe His
Arg Arg Ile Lys Ala1 5 10 578PRTArtificial SequenceSynthetic
Peptide 57Cys Gly Gly Asp Gly Glu Ala Gly1 5 5815PRTArtificial
SequenceSynthetic Peptide 58Cys Gly Gly Asn Gly Glu Pro Arg Gly Glu
Thr Tyr Arg Ala Tyr1 5 10 15 595PRTArtificial SequenceSynthetic
Peptide 59Ile Lys Val Ala Val1 5 6016PRTArtificial
SequenceSynthetic Peptide 60Asp His Lys Phe Gly Leu Val Met Leu Asn
Lys Tyr Ala Tyr Ala Gly1 5 10 15 6113PRTArtificial
SequenceSynthetic Peptide 61Gly Gly Cys Arg Trp Tyr Ala Lys Trp Val
Cys Val Trp1 5 10 6213PRTArtificial SequenceSynthetic Peptide 62Ser
Lys Cys Trp Gly Trp Thr Pro Tyr Tyr Cys Val Ala1 5 10
6313PRTArtificial SequenceSynthetic Peptide 63Val Trp Cys Gly Met
Phe Gly Lys Arg Arg Cys Val Thr1 5 10 6413PRTArtificial
SequenceSynthetic Peptide 64Trp Asn Cys Ile Lys Gly Ser Ser Trp Ala
Cys Val Trp1 5 10 6513PRTArtificial SequenceSynthetic Peptide 65Trp
Tyr Cys Phe Arg Glu Asn Lys Tyr Val Cys Val Met1 5 10
6613PRTArtificial SequenceSynthetic Peptide 66Trp Ser Cys Pro Lys
Val Asn Gln Tyr Ala Cys Phe Trp1 5 10 6713PRTArtificial
SequenceSynthetic Peptide 67Trp Val Cys Leu Trp Arg His Arg Gly Asp
Cys Ser Ile1 5 10 6813PRTArtificial SequenceSynthetic Peptide 68Ser
Leu Cys Ala Ala Tyr Asn Arg Trp Ala Cys Ile Trp1 5 10
6913PRTArtificial SequenceSynthetic Peptide 69Trp Arg Cys Leu Gly
Asp Gly Tyr His Ala Cys Val Arg1 5 10 7013PRTArtificial
SequenceSynthetic Peptide 70Leu Glu Cys Pro Gly Glu Ser Lys Tyr Tyr
Cys Ile Tyr1 5 10 7113PRTArtificial SequenceSynthetic Peptide 71Trp
Glu Cys Ala Glu Glu Ser Lys Phe Trp Cys Val Phe1 5 10
7213PRTArtificial SequenceSynthetic Peptide 72Trp Phe Cys Leu Leu
Gly Arg Ser Ala Tyr Cys Val Arg1 5 10 7313PRTArtificial
SequenceSynthetic Peptide 73Gln Gly Cys Ala Phe Val Thr Tyr Trp Ala
Cys Ile Phe1 5 10 7413PRTArtificial SequenceSynthetic Peptide 74Lys
Leu Cys Cys Phe Asp Lys Gly Tyr Tyr Cys Met Arg1 5 10
7513PRTArtificial SequenceSynthetic Peptide 75Trp Trp Cys Lys Lys
Pro Glu Tyr Trp Tyr Cys Ile Trp1 5 10 7613PRTArtificial
SequenceSynthetic Peptide 76Leu Val Cys Asn Arg Gln Asn Pro Trp Val
Cys Tyr Ile1 5 10 7713PRTArtificial SequenceSynthetic Peptide 77Trp
Val Cys Asn Asp Leu Ile His His Phe Cys Val Trp1 5 10
7813PRTArtificial SequenceSynthetic Peptide 78Arg Leu Cys Cys Trp
Lys Thr Gln Tyr Phe Cys Glu Ile1 5 10 7913PRTArtificial
SequenceSynthetic Peptide 79Met Tyr Cys Glu Arg Asp Ser Lys Tyr Trp
Cys Ile His1 5 10 8015PRTArtificial SequenceSynthetic Peptide 80Arg
Arg Glu Leu Val Arg Met Thr Asp Trp Val Trp Val Ser Gly1 5 10 15
8115PRTArtificial SequenceSynthetic Peptide 81Leu Glu Asp Ala Met
Gly Trp Ala Leu Ser Trp Gly His Ile Trp1 5 10 15 8213PRTArtificial
SequenceSynthetic Peptide 82Trp Leu Cys Leu Asp Lys Asn Cys Met Ala
Cys Val Trp1 5 10 8313PRTArtificial SequenceSynthetic Peptide 83Trp
Leu Cys Lys Gly Ser Asn Lys Tyr Met Cys Glu Trp1 5 10
8413PRTArtificial SequenceSynthetic Peptide 84Trp Asp Cys Gly Lys
Lys Asn Ala Trp Met Cys Ile Trp1 5 10 8513PRTArtificial
SequenceSynthetic Peptide 85Trp Val Cys Ile Trp Glu Arg Phe Lys Ser
Cys Asn Glu1 5 10 8613PRTArtificial SequenceSynthetic Peptide 86Ile
Trp Cys Gly Ser Arg Phe Gly Cys Trp Cys Lys Pro1 5 10
8715PRTArtificial SequenceSynthetic Peptide 87Gly Phe Val Leu Val
Trp Ser Tyr Thr Cys Arg Cys Trp Gly Lys1 5 10 15 8813PRTArtificial
SequenceSynthetic Peptide 88Ser Thr Cys Ser Trp Val Ser Ser Tyr Val
Cys Ile Met1 5 10 8913PRTArtificial SequenceSynthetic Peptide 89Phe
Trp Cys Ile Arg Gly Glu Tyr Trp Val Cys Asp Arg1 5 10
9015PRTArtificial SequenceSynthetic Peptide 90Ser Asp Trp Ser Val
Leu Leu Ser Cys Glu Arg Trp Tyr Cys Ile1 5 10 15 9115PRTArtificial
SequenceSynthetic Peptide 91Glu Ser Gly Leu Lys Val Met Cys Met Lys
Tyr Tyr Cys Met Ala1 5 10 15 9213PRTArtificial SequenceSynthetic
Peptide 92Tyr Met Cys Met Ser Arg Gly Asp Ala Thr Cys Asp Val1 5 10
9313PRTArtificial SequenceSynthetic Peptide 93Gly Glu Cys Phe Tyr
Tyr Val Met Asn Thr Cys Val Trp1 5 10 9413PRTArtificial
SequenceSynthetic Peptide 94Trp Trp Cys Asp Met Arg Gly Asp Ser Arg
Cys Ser Gly1 5 10 9515PRTArtificial SequenceSynthetic Peptide 95Trp
Thr Trp Glu Ser Ala Phe Ala Gly Arg Trp Glu Val Gly Asp1 5 10 15
9613PRTArtificial SequenceSynthetic Peptide 96Glu Ser Cys Trp Tyr
Gln Ile Met Tyr Lys Cys Ala Asn1 5 10 9711PRTArtificial
SequenceSynthetic Peptide 97Leu Asn Cys Ala Met Tyr Asn Ala Cys Ile
Trp1 5 10 9813PRTArtificial SequenceSynthetic Peptide 98Gln Cys Cys
Gln Leu Arg Gly Asp Ala Val Cys Asn Cys1 5 10 9913PRTArtificial
SequenceSynthetic Peptide 99Trp Gln Cys Gly Arg Phe Trp Cys Ile His
Cys Leu Trp1 5 10 10013PRTArtificial SequenceSynthetic Peptide
100Leu Glu Cys Thr Glu Arg Gly Asp Phe Asn Cys Phe Val1 5 10
10113PRTArtificial SequenceSynthetic Peptide 101Trp Met Cys Ser Gly
Val Gln Pro Asn Ala Cys Val Trp1 5 10 10213PRTArtificial
SequenceSynthetic Peptide 102Leu Cys Cys Glu Ser Tyr Ile Cys Ala
Leu Cys His Tyr1 5 10 10315PRTArtificial SequenceSynthetic Peptide
103Asp Leu Cys Thr Tyr Gly His Leu Trp Leu Gly Asn Gly Arg Pro1 5
10 15
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